Interactions of Herpes Simplex Virus Type 1 Thymidine Kinase and the Peripla
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Thermodinamic characterization of protein/ligand-interactions of herpes simplex virus type 1 thymidine kinase and the periplasmic domain of the histidine autokinase CitA Author(s): Perozzo, Remo Publication Date: 1999 Permanent Link: https://doi.org/10.3929/ethz-a-003886133
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ETH Library
Dissertation ETH No. 13350
Thermodynamic Characterization of Proteîn/LigancfInteractions of Herpes Simplex Virus Type 1 Thymidine Kinase and the Periplasmic Domain of the Histidine Autokinase CitA
A dissertation submitted to the
Technology Zurich degree of
Swiss Federal Institute of for the
Doctor of Natural Sciences
presented by Remo Perozzo Pharmacist
(Eidg, dipl. Apotheker) ETH Zurich
born November
19th,
1968
citizen of Zurich, Switzerland
accepted
on
the recommendation of
Prof. Dr. G. Folkers, examiner Dr. L
Scapozza,
Dr. I.
Jelesarov, co-examiner
1999
co-examiner
Table of Contents
Table of Contents
!
Table of Contents
IV
Abbreviations
VII
Summary
XI
Zusammenfassung
PART 1
.
.
1
isothermal Titration Calorimetry 1.1. Introduction to 1.2. Historical
7
Principles and Properties
1.4. Isothermal Titration
9
Calorimetry
12
Experimental Design
1.6. Data 1.7.
3
Background
1.3. Calorimetric
1.5.
3
Calorimetry
17
Analysis
Strategies for Measuring Tight Binding Affinity
26
1.8. Informational Content of ITC Data 1.9. Prediction of
1.10.
31
Binding Energetics
Thermodynamics
and Rational
22
Drug Design
39 41
1.11. References
Herpes Virus Type 1 Thymidine Kinase 2.1.
Herpes
51
Viruses
52
Thymidine Kinases Structure and Function of HSV1 Medicinal
Aspects of HSV1 TK
References
Thymidine
Kinase
55
62 64
Table of Contents
Il
Periplasmic
Histîdine Autokinase CîtA 73
3.1. Citrate Metabolism 3.2.
Regulation of Citrate
75
Fermentation
77
3.3. References
Aims and 4.1.
Scope
of the Presented Work 79
Objectives
4.2. References
81
PART 2
83
Method Development and
Improvement 85
1.1. introduction 1.2.
Expression
86
and Purification
91
1.3. Concentration Determination Procedures 1.4. Kinetic and
91
Activity Measurements
1.5.
Stability Measurements
94
1.6.
High Performance Liquid Chromatography
96 99
1.7. References
101
PART 3
Compulsory HSV1 1.1.
Order of Substrate
Thymidine
Summary
Binding
Kinase: A Calorimetric
to
Study 105
1.2. Introduction
106
1.3. Results
109
1.4. Discussion
119
1.5. Conclusion
123
1.6. Material and Methods
124
1.7. References
126
Table of Contents
Substrate Diversity of Herpes Simplex Virus Thymidine Kinase
-
Impact of the Kinematics of the Enzyme 2.1.
135
Summary
135
2.2. Introduction
Results
143
Discussion
148
References
153
The
Periplasmic Functions
3.1.
138
Procedures
Experimental
Domain of the Histidine Autokinase CitA
as a
Highly Specific Citrate Receptor 160
Summary
3.2. Introduction
160
3.3. Results
163
3.4. Discussion
172
3.5.
Experimental
180
Procedures
3.6. References
186
PART 4
191
Appendix 1.1. List of Publications
193
1.2. Posters
193
1.3. Oral Presentations
194
1.4. Curriculum Vitae
195
IV
Abbreviations
Abbreviations
ACV
acyclovir, 9~(2-hydroxyethoxymethyl)guanine
ADK
adenylate
ADP
adenosine
ADPCP
ß,y-methyleneadenosine 5'-triphosphate
AMP
adenosine
AMPCF2P
ß,y-difluoromethyleneadenosine 5'-triphosphate
Ap5T
P
ATP
adenosine
AZT
3'~azido-3'-deoxythymidine
CD
circular dichroism
ACp
heat
AG
free energy
AH
enthalpy change
AS
entropy change
dC
2'-deoxycytidine
dCK
deoxycytidine
dT
thymidine
dTMP
thymidine 5'-monophosphate, thymidylate
dTDP
thymidine 5'-diphosphate
dTTP
thymidine 5'-triphosphate
DEAE
diethylaminoethyl
DFT
density functional theory
DTT
D/L-dithiothreitol
EDTA
ethylenediamine
E. coli
Escherichia coli
GSH
glutathione
GST
glutathione S-transferase
GST-TK
fusion
HPLC
high performance liquid chromatography
HSV1
herpes simplex
kinase
5Ldiphosphate
5'-monophosphate
5
1
-(adenosine-S')-P -(thymidine-5')-pentaphosphate
5'-triphosphate
capacity change
change
protein
kinase
of
tetraacetic acid
glutathione S-transferase
virus type 1
and HSV1 TK
V
Abbreviations
HSV1 TK
herpes simplex
hTK1
human
IPTG
isopropyl-ß-D-thiogalactopyranoside
ITC
isothermal titration
Km
Michaelis-Menten constant
LDH
lactate
NADH
nicotinamide-adenine dinucleotide, reduced form
PAGE
Polyacrylamide gel electrophoresis
PCR
polymerase
PK
pyruvate kinase
PEP
phosphoenol pyruvate
PMSF
phenylmethylsulfonyl fluoride
SDS
sodium
TmpK
thymidylate kinase
TK
thymidine kinase
TK"
TK
Tris
tris(hydroxymethyl)aminomethane
"max
virus type 1
cytosolic thymidine
thymidine
kinase
kinase
calorimetry
dehydrogenase
chain reaction
dodecyl sulfate
negative
rate of enzyme
catalyzed
reaction at infinite concentration of substrate
The one-letter code is used for amino acids.
/
,
V I
fjjua.1*
Summary
VII
Summary Understanding of
molecular
ligand binding
receptor and
binding,
complete characterization data with
interacting
of the
processes
binding energetics
associations
in
substrate
and correlation of
of
determination
free energy of
binding
fundamental
strategies.
of
is
process
significant
for
know-how
of
calorimetry (ITC)
interactions in terms of
a
all
binding (AG), enthalpy (AH)
and
virus
forming
the
second
focus
type
1
close
a
it
discusses
thymidine
kinase
catalytically competent of this work is the
insight
into
provides the
design
molecular
premier
tool for
and elucidation of
binding
emerged
as
the
investigation
thermodynamic point of view, exemplified for
systems. The first system
simplex
thermodynamic
thermodynamic parameters.
The main aspect of the present work is the characteristics from
a
of
structure-based has
requires
of the forces that
practical interest, since
development
Isothermal titration
characterizing
and
inhibitor
changes
entropy (AS) of binding and the heat capacity change (ACp). Such the
and
interactions
quantitative description
include
thermodynamic parameters, including
as
protein-protein
or
structures involved. A
molecular
govern
recognition
the
ligand-enzyme
(HSV1 TK)
with
As for
state.
two different
interactions
respect
to all
of
herpes
elementary steps
ligand-receptor interactions,
investigation of
the
properties of the
the sensory
periplasmic domain (CitAP) of the histidine autokinase CitA. Thymidine kinase is
y-phosphate to
form
target gene
a
transfer from ATP to
in the
pyrimidine salvage pathway catalyzing the
thymidine (dT) in presence of magnesium cations
thymidine monophosphate (dTMP). HSV1
in medicinal
therapy of
properties
chemistry because of
of HSV1 TK is
HSV1 TK
were
proteins using
as
rational
therapy.
The
thrombin cleavable
E. coli. BL21 as the host.
purification
knowledge
drug design wild-type
yield high
of
binding
and effective
and mutant enzymes of
glutathione S-transferase
(affinity
amounts of active and
important
of the
new
Activity studies and kinetic
protocol
an
therapy of viral infections and
stability of the fusion protein compared
single-step
established to
its links with
prerequisite for
and antitumor
expressed
showed enhanced a
a
become
TK has
and AIDS. Thus, the accurate
cancer
compounds for antviral
Thus,
key enzyme
ligand
measurements
to cleaved
chromatography) free fusion
fusion
HSV1 TK. has
been
protein. Conditions
VIII
Summary
for ITC measurements
possible systematic
and verified in order to avoid
subsequently optimized
were
errors.
ITC has been used to
investigate
the
binding energetics
and natural cofactor ATP to HSV1 TK, either in isolation
ligand,
range of
in the temperature
10-25°C.
of the natural substrate dT in presence of the other
or
The results of the
measurements show that dT is bound in the micromolar range
Under the
same
conditions
binary complexes and 0.26 uM,
a
dramatic increase of
contributions
are
range. The temperature dependent in the range of 140 to 520
and
negative
both dT and ATP
over
capacity changes
calK^mor1.
the
to
entropy changes
known
by structure-based
of the ternary HSV1 j^.^j.^jp
structure
indicate
linked
were
well
as
the
as
substrate and cofactor. In
the LID
analogy
binding
harboring
to
closed
a
approach.
distinguishable from
compact
by
more
other
mutant H58L7M128F/Y172F
one.
of
exhibiting
Calorimetric
studies
binding
of
even
positive entropy
similar values
importance
of the
structural rearrangement of substrate and cofactor
length and
is
large
HSV1 TK
strength
as
composed
of
(131 amino acids), cytoplasmic domain, and
a
an
to
mechanisms.
of
of
not
lose
Affinity
enthalpic with the
binding. The triple
which is also mirrored in
for the wild type enzyme.
interplay
binding
were
shown
with much less favorable
regains phosphorylation activity
the
a
propose
(M128F, M128F/Y172F) that
magnitude
thermodynamic parameters revealing
CitA is 547 amino acids in
binding
entropie contributions become less unfavorable,
double mutant M128F/Y172F
further corroborates
is
empty (open) and less ordered
an
due to extensive alterations in
contributions. In contrast,
domain
we
by kinetic measurements, have
than two orders of
which
smaller but still substantial movement of
Inactive mutants
each
phosphorylation activity is reduced
and
kinases,
to
mutations in the residue triad H58/M128/Y172 demonstrate the
the calorimetric
This
a
domain, with the protein going from
conformation
the
to other nucleotide
domain and
observed
sequential binding pathway,
a
possibly realized by structural rearrangements of the enzyme coupled
movement of the dT
(46 nM
the whole temperature
revealed heat
binding enthalpy
complex. The pronounced changes in heat capacity unfavorable
uM; pH 7.5).
by favorable enthalpy changes
driven
are
large
affinity of
Experimentally obtained thermodynamic quantities thermodynamic analysis
5.3
of ATP is detectable. The measurement of the
Associations
respectively).
entropie
while the
showed
binding
no
(KD
thermodynamic
between
binding
and
of HSV1 TK. aminoterminal
periplasmic
carboxyterminal cytoplasmic
Summary kinase domain kinase of the
harboring
the
autophoshorylable histidine
the induction of
responsible
was
periplasmic domain
C-terminally
HSV1 TK,
with
a
provided in form of the recombinantely overproduced
optimal ITC conditions
(CitAPms). Similarly
to
binding characteristics of CitAPHis-
neither
contrast,
isocitrate
Association is driven by
large
negative.
and
dianionic form
is not bound
of CitA is This
work
processes of small
in rational
pM, pH 7)
is the
a
1:1
stoichiometry.
binding.
the
recognized species
binding .
reaction indicated that the
In the presence of
Mg2+
ions the
that the
Mg-citrate complex
The result of this work reveals that the
periplasmic domain
ligand
citrate receptor.
ITC
that
and
binding
drug design.
in
tricarballylate exhibit demonstrable
pH-dependence of
is
valuable
biological
thermodynamic data is large,
in the elucidation of
nor
5
favorable enthalpy change while the entropie contribution
highly specific
confirms
high affinity (KD
significantly increased, suggesting
by CitAPHis.
as a
a
The
H-citrate2~
dissociation constant
of
attached histidine tag
have been established for this system to elucidates
Purified CitAPHiS binds citrate with
is
two-component regulatory system
in presence of citrate.
For this work, the receptor
In
sensor
enzymes involved in citrate fermentation
under anaerobic conditions. The activation of the
the
residue. It is the
two-component regulatory system CitAB of K. pneumoniae, which is
responsible for
occurs
IX
and it is
method
for
characterizing recognition
macromolecules. The informational content
anticipated that
mechanisms and,
through
it will
play
an
important role
the link to structural data, also
Zusammenfassung
XI
Zusammenfassung Die
Beschreibung
von
molekularen
von
Substraten
und
Inhibitoren,
Erkennungsprozessen,
wie sie bei der
Rezeptor/Ligand-
bei
Bindung
Protein/Protein-
oder
Wechselwirkungen stattfinden, bedarf der umfassenden Charakterisierung aller
Bindung beitragenden
(ACp).
Wärmekapazität
der
Die
dieser
Entropie (AS)
Daten
In
Design.
(ITC)
Zusammenhang
diesem
als Methode erster Wahl
Wechselwirkungen
zur
zur
auf der Basis
von
Hauptinteresse der vorliegenden
von
Bindungseigenschaften
zwei verschiedenen
Wechselwirkungen aller
elementaren
Systemen.
der
führen. Als Beispiel für Domäne
(CitAP)
Thymidinkinase
ist das
Erkrankungen
medizinische
des
die
HSV1 TK
zu
auf
sowie
zur
Aufklärung an
Enzym/Ligand-
(HSV1 TK) bezüglich
1
katalytisch
kompetenten
dient die
Komplex
periplasmatische
deren sensorischen
der
Zwischen
in
Eigenschaften
von
endständigen Phosphatrestes
von
HSV1 TK
Enzym ist.
Wiederverwertung
Gegenwart
Gentherapie
weshalb das
um
metabolischen
des
Thymidin (dT)
Forschung geworden
erhalten,
und die
befasst sich mit
Herpesvirus Typ zum
grossem Interesse, präzise und detaillierte von
molekularen
von
Parametern etabliert.
Untersuchung
System
Übertragung
entsteht.
Zusammenhang gefunden, die
Beschreibung
Tricarballylat untersucht worden ist.
wobei durch die
Thymidinmonophosphat viralen
Ein erstes
Schlüsselenzym
Adenosintriphosphat (ATP)
Drug
Rational
zum
thermodynamischen Blickwinkel, gezeigt
der Histidin-Autokinase CitA, Isocitrat und
Pyrimidinen,
und
Rezeptor/Ligand-Interaktionen
bezüglich Citrat,
beteiligten
Isothermale Titrationskalorimetrie
Arbeit ist die
Thymidinkinase
und
Bindungsprozesse
Strategien
thermodynamischen
einem
Bindungsschritte,
von
hat sich die
Bestimmung
Das
aus
Entwicklung
den
mit
Strukturen auf molekularer Ebene bietet einen tiefen Einblick in
und liefert fundamentales Wissen
Änderungen
mit ein:
der
Bindungsenthalpie (AH),
Korrelation
dirigierenden Kräfte
der
thermodynamischer Parameter
Bindungsenergie (AG),
der freien
der
aller
Bestimmung
schliesst die
quantitative Beschreibung
Kräfte. Die
zur
von
zu
von
der
und
Krebs
und
Behandlung AIDS
einem interessanten
Unter diesen
Angaben
mittels rationalem
Magnesiumkationen
über die
von
ein
wurde
Zielobjekt
Umständen
ist
es
für
von
Bindungseigenschaften
Wirkstoffdesign
Wirkstoffe für die antivirale und antineoplastische Therapie
zu
neue
und effiziente
entwickeln.
Dazu
XII
Zusammenfassung
wurden
Wildtyp
und Mutanten der HSV1 TK in Form des Glutathion S-Transferase-
Fusionsproteins exprimiert eine
Messungen einstufige Reinigung
und
isolierung
von
aktivem
ATP
auf
wurden
kinetische wurde
eine
Affinitätschromatographie,
zur
fortlaufend
etabliert.
und
optimiert
mittels
systematische Fehler untersucht.
HSV1 TK
an
und
ligandfreiem Fusionsprotein
und
des natürlichen Substrates dT und
Bindungseigenschaften
Mittels ITC wurden die
Aktivitätsstudien
Fusionsprotein zeigten,
basierend
Messbedingungen
verschiedener Methoden auf
Kofaktors
Da
Stabilität für das
bessere
Reinigungsprozedur,
Kalorimetrische
des
gereinigt.
und
im
Temperaturbereich
10°C
von
bis
25°C
untersucht, jeweils mit ligandfreiem Enzym und mit den korrespondierenden binären
Mischungen.
Die Resultate
7.5) bindet,
während
detektiert werden
kann.
alle
entropischen
Die
(46
binären
Versuchen als stark
von
dominiert.
keine
(KD
Im untersuchten
|jM).
Enthalpiewerte
temperaturabhängig
stark erhöhte
und
erwiesen
ACp
mit Werten für
ATP
von
Temperaturbereich
günstigen enthalpischen
Die
uM; pH
5.3
Bindung
Mischungen hingegen zeigen
nM bzw. 0.26
Wechselwirkungen Beiträgen
dass dT im mikromolaren Bereich
gleichen Bedingungen
den
unter
Affinität für dT und ATP werden
zeigen,
ungünstigen
sich
in
allen
zwischen -140 bis -510
calK~1mor1. Die
Korrelation
von
HSV1 TK:dT:ATP
Wärmekapazität auf
einen
experimentellen Daten
Komplexes lässt den Schluss
und der
Entropie
des
Enzyms
Nukleotidkinasen
geht
die
in
geordneten
eine
Konformationsänderung
Anhand
bei der
Bindung
von
eine
von
geschlossene,
wird durch eine grosse
H58/M128/Y172 konnte die
Studien
sind.
In
von
massive
und
für
Bindungsmechanismus
offenen
in
in der
jede
der
signifikante zu
anderen
und
weniger
über.
Diese
NMPbind-Domäne
der LID-Domäne verursacht.
Mutanten
der
Aminosäuren-Triade
Bedeutung des thermodynamischen
Messungen
eine
Konformation
werden. Der Aktivitätsverlust der inaktiven Mutanten sich durch kinetischen
auf
Analogie
leeren,
Bewegung
weniger ausgedehnte Bewegung
kalorimetrischen
auch
kompakte
ternären
Substrat und Kofaktor sowohl
als
einer
von
des
Änderungen
zu, dass die starken
zurückzuführen
HSV1 TK
Kristallstruktur
der
sequentiellen Bindungsmechanismus,
Strukturänderung
und durch
mit
Ansatzes
gezeigt
(M128F; M128F/Y172F),
die
nicht voneinander unterscheiden lassen, ist auf
Mutante
unterschiedliche
zurückzuführen. Die Affinität ist
um
zwei
Änderungen
des
Grössenordnungen
XIII
Zusammenfassung
günstigen enthalpische Beiträge
reduziert und die
aber
ungünstige
der
M128F/Y172F
Beitrag
entropische
zeigt
und
geringer,
die
Einfluss.
positiven
einen
sogar
sind stark reduziert.
Insgesamt
ist
Doppelmutante
Tripelmutante
Die
H58L/M128F/Y172F erlangt die Aktivität zurück. Das widerspiegelt sich auch in den
thermodynamischen Parametern, werden doch ähnliche
gefunden.
Beobachtungen
Diese
Zusammenspiels
zeigen
Werte wie für den
nochmals
Wildtyp
Wichtigkeit
die
des
Bindung und strukturellen Anpassungen bei der Interaktion
von
von
Substrat und Kofaktor mit HSV1 TK. CitA ist ein 547 Aminosäuren
periplasmatischen und einer
bestehend
langes Protein,
einer aminoterminalen
aus
(131 Aminosäuren), einer cytoplasmatischen Domäne,
Domäne
carboxyterminalen Kinase-Domäne,
die das
autophosphorylierbare
Histidin
trägt. CitA ist die Sensorkinase des Zweikomponenten-Regulationssystems CitAB von
K.
pneumoniae,
anaeroben
das für die Induktion der
Bedingungen
Enzyme des Citrat-Stoffwechsels
verantwortlich ist, wobei die
Aktivierung
Für die
vorliegende
und mit einem
Arbeit wurde der
in Form der rekombinant
Rezeptor
Polyhistidinrest modifizierten periplasmatischen
eingesetzt. Analog
HSV1 TK wurde ein
zu
Bindungseigenschaften Gereinigtes CitAPHis
von
mittels ITC untersuchen
gleichen Bedingungen
(KD
binden
Citratanaloge
unvorteilhafte
aus.
Komplex
nicht
an
von
hochspezifischer Citrat-Rezeptor
Diese
Arbeit
zeigt deutlich,
Wechselwirkungsprozessen Makromolekülen ist.
angenommen
wie von
erkannt
wenn
es
rationales Wirkstoff design
dass
wertvoll
um
geht.
die
die
kleinen
die
und
und
wird
es
und von
konnte
der
ist
gezeigt
Mg:Citrat-
CitA kann somit
bezeichnet werden.
Thermodynamische werden,
wie Isocitrat und
Magnesiumkationen
pH-abhängig,
H-Citrat2"
die
um
günstige enthalpische
CitAPHiS bindet. Die periplasmatische Domäne
als ein
einnehmen wird,
Gegenwart
In
reduziert. Die Affinität ist
werden, dass die dianionische Form
(CitAPms)
und mit einer 1:1
jjM; pH 7)
5
zeichnet sich durch
Bindung signifikant
Domäne
können.
zu
Tricarballylat nicht. Die Interaktion entropische Beiträge
exprimierten
experimentelles System etabliert,
bindet Citrat mit hoher Affinität
Stöchiometrie. Unter
darf
Gegenwart
in
erfolgt.
Citrat
die
unter
ITC
für
die
Charakterisierung
Ligandmolekülen
mit
von
biologischen
Daten liefern viele Informationen, und ITC
in
Aufklärung
Zukunft
von
einen
hohen
es
Stellenwert
Bindungsmechanismen
und
um
PARTI
PARTI
Introductions
1
£.*'
\
>
Calorimetry
Isothermal Titration
1.1. Introduction to
macromolecules
recognition. Biological with
large molecules, and
prerequisite
for
interactions
is
Calorimetry
principle of all biological processes is molecular organization and
A fundamental
chemists
a
high degree
biologists from
a
the
thorough
The
formation.
by measuring
subject of
a
very
and with
high affinity, fascinating
modern
of
beginning
Calorimetry is
heat
and
biochemistry.
the
quantification of
quantities
or
the
only technique enabling
between and within
A
a
energetics us to
study
macromolecule in sufficient
heat effects.
Background of the development of
background
specificity
characterization
directly the basic physical forces
1.2. Historical
of
able to interact with various small and
are
deeper understanding of the molecular basis of protein-ligand
a
governing complex
detail
3
Calorimetry
PART 1: Isothermal Titration
variety
calorimetry and thermodynamic
of historical studies, and this
summary of the most
interesting aspects
thereof
chapter is
(Cobb
has been the
meant to
be
a
short
& Goldwhite, 1995; Daumas,
1950; Guerlac, 1976; Hemminger& Höhne, 1979; Hudson, 1992; Lodwig & Smeaton,
1974; Partington, 1989; Pledge, 1939; Wintermeyer, 1974).
Calorimetry
is
calorimetry
and
a
very
old
science.
thermodynamic
In
began
principle, with
the
the
historical
description
development
of
definition
of
and
temperature and heat. The first known documents from the early 17th century witness for very crude attempts to describe temperature, most of them derived
"heat of
a
estimations
breeding hen, were
too
heat of
rough,
by perception:
boiling water, heat of glowing charcoal". These
and therefore it
was
necessary to
develop objective
PART 1: Isothermal Titration
4
Calorimetry
The concept of
period.
known from used
antiquity
bulb with
a
an
expansion of gases and liquids due
and
thermoscops
were
with the effects of barometric pressure and lacked of
satisfactory
thermometers
ethanol, mercury) in
temperature scale
a
was
established
time when the nature of heat and it
speculated
had
People
the
on
widespread belief that heat of atoms.
by Joseph
Black. He
can
He
be absorbed
recognized
mixture but
or
preferred
squeezed
that heat
was
by
of
a
18th century
to
melting
between the
of
ice,
covered
reliable
a
ice did not
change
plate of
ice
It
times.
calorimetry
heat
being
was
it
a
was
were
a
place
laid
fluid that
to another.
temperature of the
the
for the first time
of heat could be estimated from
him to first calorimetric
warm
around this
was
"amount" of heat. Black introduced
quantities
brought
a
the view that
held
some
It
solid-liquid phase transition,
calorimeter. A
with
17th century
ancient
since
explanation of
"strength" and
the amount of melted ice. This view
block
(water,
became of interest.
the foundations of
alternative
an
instead of air
out of bodies and can flow from one
applied
simple phase-transition
heat
substance,
the concept of latent heat and showed that
a
(pure) liquids
Fahrenheit and Celsius.
nature
consumed for the
clearly discriminating
of
use
The next crucial step to
scaling.
quantitative aspects
s
was
the
During
composed
the
expansion
still very inaccurate, confused
closed compartment. Until the end of the
was
already
was
stem inverted over water to observe the
open-ended
of air. The results of these so-called
make
heat
to
by Galileo and Drebbel. They independently
used
was
in the same time
origin
standards. The invention of the first thermometer had its
probe and
was
placed
brought
to
experiments with
in the
thermal
cavity of
an
equilibrium.
Furthermore, he adopted the idea of mixing water of different temperatures (mixing
calorimeter) from Brooke Tylor (1723)
to
determine
a
series
of
latent
heats
of
different substances. At the
heat.
same
time, A.L. Lavoisier and P.S. Laplace became interested in the theory of
They considered the widespread mixing calorimeters
several
vessel
disadvantages: and
inmiscible
produced
the need
thermometer,
heat
liquids. Moreover,
during
as
unsuitable because of
of delicate corrections for the heat
loss
capacity of
by cooling, chemically reacting substances,
this method did not allow the measurement of the heat
combustion
and
other
chemical
reactions,
and
during
Calorimetry
PART 1: Isothermal Titration
Fig.1
:
The ice caiorimeter of Lavoisier and
Laplace (from Oeuvres
5
Lavoisier, Tome Premier, Paris,
de
Imprimerie Impériale, 1862)
in which
respiration, topics convenient
were
mostly interested. They developed the first
phase transition calorimeter that led
simple but ingenious
respiration
and
surrounded
by
another
they
ice calorimeter, a device for
combustion
an
ice-packed jacket,
ice-packed jacket
to
the melted ice of the inner chamber. The
(Fig.1).
handling
measuring
instrument
was
used
difficult and
days when the outside temperature
was
was
It
was
a
heat release due to
of
a
chamber
further insulated with
amount of water collected from
as a measure
experiments a
results.
consisted
and the whole device
improve accuracy. The
jacket
was
The
reproducing
to
of the heat evolved in the
could
only
few degrees above
be
performed
freezing.
on
With this
device, Lavoisier and Laplace determined the specific heat of various substances and found
fairly good
experiments heat
were
during
a
guinea pig
the
to
modem standards. The most famous
respiration
Laplace
measured the
and determined the amount of carbon dioxide in its
experiment. They compared
carbon dioxide formation when to conclude that
compared
conducted about 1780, when Lavoisier and
generated by
exhaled air
results
burning
was a
it to the heat release and to
charcoal. The results
form of combustion.
were accurate
enough
6
PART 1: Isothermal Titration
Despite
this
interesting experimental work,
the
Calorimetry
resulting interpretations of
of heat remained unclear. Lavoisier still treated heat called it caloric, matter of fire.
Laplace favored
motion of
view that
out
of
particles of matter,
experimental
a cannon was
large quantity
a
bored and that there
was no
produced by simple drilling. He concluded that heat caloric), otherwise it had was a
to
stop when the
big controversy about this theory,
century when the caloric theory
was
of heat
was
as
generated
limit of amount that could be
motion and not matter
was
cannon was run out
(or
of caloric. But there
and it was not until the middle of the
finally
as
18th century
toward the end of the
emerged
Rumford noticed that
being
explanation of heat
provided by Count Rumford, formerly known
evidence
Benjamin Thompson. when
a
weightless substance and
as a
mechanical
a
the nature
overthrown. The kinetic gas
theory
18th was
established and the concept of energy arose. With the Industrial Revolution
became
of
more
combustion could
than
beginning
academic
produce work,
concerned with the rules
Calorimetric
With
be it that
biologists
outside
physical, chemical calorimetry
particular
in
expression
could
remained
is
more or
accurately very Since
the
small heat
middle
of
less the
the
from
was
born.
It is
same
was
an
small
during this
time
improvements. Until the last few
accompanied by
practically
heat
tool for
a
that made available
increasing
thermodynamic data. This gave the imputs
requiring only
heat
to become of interest to biochemists
powerful analytical
purification techniques, there
that
of energy and is able to
to
develop
sample quantities
molecular
significant
new
and
it is obvious
variety of applications,
for
need
every process,
changes,
sciences. With concurrent advances in
homogeneous protein,
calorimeters,
thermodynamics
modifications and
biological,
serve as
biological
and
realization
few specialized laboratories. Since or
the nature of matter
processes.
were some
a
the
the interconversion
decades, calorimetric techniques have started and
19th century,
the science of
measuring techniques
period, although there
interest.
governing
predict the feasibility of chemical
in the
more
biology,
amounts of
and
reliable
and very sensitive
being
able
to
detect
quantities.
20th century
several
calorimetric
principles
of
different
practical design have emerged. But it is only since the last few years, with the
development
and
improvement of sufficiently sensitive, stable,
affordable commercial calorimeters, that allowed
calorimetry
user
friendly
to become
an
and
almost
PART 1: Isothermal Titration
routine
analytical procedure
instruments
very sensitive,
are
requiring only
1.3. Calorimetric
Principles
there
is
principles of calorimetry the
areas:
construction
sample
in
a
biophysical
changes
7
research. Since modern
in the range of
volume of 0.2 to 1.4 ml,
microcalories,
they
are
usually
microcalorimeters.
as
Unfortunately,
heat
detecting
10 to 100 nmol of
denominated
important
in biochemical and
Calorimetry
and
rather
a
Properties
confusing
and calorimeters. In
measuring principle,
(Hemminger
&
collection
general,
the
of
describing
names
it is useful to
operating mode,
the
separate three
and
the type of
Höhne, 1979; Oscarson & Izatt, 1992; Wadsö, 1975;
Wadsö, 1994).
1.3.1. Measurement
Calorimeters measurement
are
Principles
instruments used to
principles
have found
quantify
use.
In
heat effects
or
heat effects. Several
principle, solution calorimeters form
two
main groups: adiabatic calorimeters and heat conduction calorimeters.
With
an
ideal
calorimeter
adiabatic
and
the
calorimeter
the heat
capacity
e
an
the
is
heat
no
heat
to the observed
exchange
between
the
quantity Q evolved during the temperature change AT, and
to
of the reaction vessel and its contents:
Q
Thus, in
and
surroundings,
experiment is directly proportional
there
=
e-AT
(1)
experiment the heat quantity is determined by measuring the temperature
change. In
an
ideal heat conduction calorimeter the heat evolved is
from the reaction vessel to the heat sink,
usually made
a
body surrounding
of metal. With this type of calorimeter,
some
the heat flow between vessel and heat sink, is measured.
recorded The
by placing
a
thermopile
temperature difference
signal
S which is
over
proportional
quantitatively transferred the calorimeter which is
property proportional to
Normally
wall between the vessel and the the
thermopile gives
rise to
to the heat flow. The time
a
the heat flow is
surrounding sink.
potential
integral for
or
voltage
the heat flow,
8
PART 1: Isothermal Titration
times
calibration constant e, is
a
proportional
Calorimetry
to the heat
quantity released in the
experiment:
Q
1.3.2.
is thus
quantity
The heat
Operating
The most
=
e-jSdt
(2)
to the area under the
proportional
signal
time
curve.
Mode
common
type of calorimeter in
use
isoperibol calorimeter,
is the
also called
"constant temperature environment" calorimeter. The vessel is separated by thermal insulation from the
surrounding
The
usually filled
insulation
is
processes will result in
practice,
there will
a
always be
very
cannot be
with
air
or
vacuum.
a
are
neglected
or
widespread
not
truly adiabatic,
in biochemical
or
as
space
thermometer. In
surrounding.
quasi-adiabatic. The heat
Isoperibol
instruments.
biological
calorimeters
They
are
used
are as
studies.
additionally
the adiabatic shield, which is
envelope,
endothermic
combustion calorimeters, but have not found
In adiabatic shield calorimeters, the reaction vessel is walled metal
but
and must be corrected for.
solution calorimeters and use
a
or
small heat loss from the vessel into the
simple and for fast processes also very precise
reaction
Exothermic
temperature change that is recorded by
Therefore, this calorimeters
exchange
thermostated bath which forms the isothermal jacket.
placed
enclosed
in the
by
vacuum
a
thin-
or
air
between the reaction vessel and the thermostated bath. The temperature
difference between the shield and the vessel is
automatically applying
1.3.3. Construction
Calorimeters
can
a
suitable heat effect
at zero
during
the
experiment by
the shield.
Design
be
in
a
arrangement is simpler, the attractive for
on
kept
single
or
a
twin
arrangement.
twin calorimeter has
microcalorimetry.
advantages
Although
the
single
which makes it very
One of the calorimetric vessels, the reaction cell,
contains the system of interest, whereas the other vessel, the reference cell, contains water or buffer. With such an
arrangement the recorded signal is
a
differential
signal,
PART 1: Isothermal Titration
9
Calorimetry
of which the effects of thermal disturbances from the
surroundings
expected
are
to
cancel out.
1.4. Isothermal Titration
Calorimetry
1.4.1. Introduction
techniques
Calorimetric
ruling
mechanisms level.
The
are
calorimetry (ITC).
differential
the
researches
have
nucleic
or
a
solution
of
constant
(KB) and thus
capacity of thermal of
stability
macromolecular
& Potekhin, 1986; Sturtevant,
biological assemblies
1987).
during molecular association. The direct a
binding event,
i.e.
a
ligand
is
the macromolecule of interest and the heat evolved
containing
enthalpy change (AH),
simultaneously determination of the equilibrium
the standard Gibbs free energy
the entropy
change (AS),
association event. Moreover, experiments the heat
and
biological
and isothermal titration
heat
about
the
at the molecular
investigate
to
learned
observable is the heat associated with
absorbed is detected. It allows the
binding
applied
and
acids)
understanding of
interacting systems
enthalpy
In contrast, ITC measures the heat evolved
titrated into
to the current
scanning calorimetry (DSC)
(Freire, 1995; Privalov, 1989; Privalov
thermodynamic
of
techniques
measures
(proteins and
macromolecule
stability
calorimetric
DSC
and
denaturation,
great impact
a
association and
principle
macromolecules
have had
as
well
performed
as
the
change (AG),
the
stoichiometry of the
at different
temperatures yield
capacity change (ACp) of the binding reaction (Chen & Wadso, 1982; Freire
era/., 1990; Wiseman era/., 1989). As almost any interacting system is characterized
by changes
in
enthalpy, there is
a
vast range of
potential ITC applications.
1.4.2. Titration Microcalorimeters
Titration calorimeters
partner into
a
are
characterized by the addition
solution of the other
of the calorimeter. It is worth very
challenging
binding partner
emphasizing
since non-covalent
the range of 5 kcal/mol to 10
(injection)
which is
that calorimetric
binding
heats
are
of
one
binding
placed in the sample cell
binding experiments
are
intrinsically small, typically
in
kcal/mol, and must be liberated stepwise during the
binding experiment. Furthermore, ligand
addition has additional heat effects
arising
10
PART 1: Isothermal Titration
from
and
dilution
mixing,
frequently comparable
Considering -5
the
kcal/mol
in
milligrams),
accuracy of
a
binding
10%
binding
typical
to
in the
containing 1Q~7
solution
made, and which
moles of
sites.
Assuming
of such
better,
sample solution of just
to the inevitable heat effects of
are
protein (a
would liberate about 0.5 meal of heat after
that this heat is released upon 10
contribution would
or
be
must
reaction of interest which exhibits the heat effect of
ml
2
corrections
heat of interest.
and noise levels down to 5
sensitivity changes
of
experiment
the
individual
mean
to the
ml
1
a
saturation of all the
case
for which
Calorimetry
be
small
jjcal a
lacal.
50
Accurate
few
complete injections,
detection, with
an
quantities would require instrumental
and less. This
few millionths of
corresponds a
degree
to
temperature
and is
comparable
dilution, mixing and stirring.
1.4.3. The OMEGA Titration Calorimeter
The
of
use
the
determination
of
calorimeter the
Recently,
measurement of the
energetics
Wadso, 1982; Freire
et
the
a
of
several
of
broad
are
insulated
by
an
accurate
for
and
direct
reported (Chen
&
al., 1984). With the introduction of the from
calorimeter
1989
(Wiseman
MicroCal
1989), titration
al.,
et
Inc.
which is reflected
by
the last decade.
This type of calorimeter consists of two cells, the that
designed
impact throughout biotechnology,
large body of publications during
and
macromolecules
to
processes have been et
in
fast
allows
microcalorimeters
titration
USA)
mode
ligand binding
biological
Omega
(Northhampton, Massachusette, calorimetry has had
titration
al., 1990; McKinnon
available
commercially
the
thermodynamics
processes.
related
in
adiabatic
shield
thermoelectric device which measures the
sample cell and the reference cell,
(Fig.2).
The
cells
are
coupled by
a
temperature difference ATi between the
cells. A second temperature difference AT2 is monitored between the two cells and the adiabatic shield. Both cells and the connected
experiment,
to a
a
feedback circuit
jacket
are
controlling AT1
small constant power
(<
1
mW)
supplied
and
is
AT2
with heaters that are
to
0.1°C/h).
The
resting
power
applied
to the
a
measurement
sample
zero.
During
an
dissipated in the heater of the
reference cell. This activates the feedback circuit to drive ATi
slowly increasing the temperature during
be
back to zero, thus
(typically
cell is the baseline
less
signal.
than
PART 1: Isothermal Titration
Calorimetry
11
Computer
Delivery syringe A
Stirrer
Regulation reference
of
Temperature
ce
sensor
Regulation 1
sample
2^ PX
ASbb
Aoex-ïu
Ala
52 1
60 4
54 6
0 00
4 11
0 00
Arg
187 9
2102
199 6
710
3 39
-0 84
Asn
1138
123 0
1129
3 30
3 39
2 25
Asp
102 4
106 5
99 4
2 01
3 39
2 15
Cys"
70 8
69 1
70 3
3 56
3 39
0 62
Cysf
91 9
94 5
92 0
3 56
3 39
0 62
Gin
128 7
142 8
122 3
5 02
3 39
2 13
Glu
117.5
135 8
124 5
3 54
3 39
2 27
Gly
00
00
00
0 00
6 50
0 00
His
144 3
£L,
144 9
3 44
3 39
0 79
lie
123 8
138 5
135 9
1 74
217
0 67
Leu
134 5
150 6
143 8
1 62
3 39
0 24
Lys
156 9
177 7
155 6
5 85
3 39
1 03
Met
158 5
160 8
158 0
4 54
3 39
0 57
Phe
1767
179 4
172 0
1 41
3 39
2 89
Pro
90 7
104 8
90 7
-
-
-
Ser
68 6
76 6
71 7
3 68
3 39
0 55
Thr
105 3
112 1
105 4
3 30
3 39
0 48
Trp
222 7
218 7
222 4
2 75
3 39
1 15
Tyr
1885
1964
190 2
2 77
3 39
3 13
Va!
105 6
1180
105 6
012
217
1 29
Î \-) £~
Values from (Baker & Murphy, 1998) Thorntoti,
are
Table 2.
Tab 2 Total side-chain ASA
ASbb, ASox-,u,
and
two additional terms associated with
and with the entropy of
state
AASAAxa,i
available, and they have been adapted for different
are
implementations (Baker & Murphy, 1998). These data For interaction processes
estimates for
,
1996),'Values
.
D(Presnell )
from
(Lee
& Richards
for disulfide-bonded cystine, Values for free
cysteine
1971)
a(Hubbard
&
PART 1: Isothermal Titration
For
non-peptide ligands
account
for
a
Calorimetry
39
special empirical parameterization
has been
proposed
changes is conformational degrees of freedom between free and
complexed forms of ligand (Bardi
et
ai, 1997). As
that the conformational entropy will be
first
a
proportional
it is assumed
approximation,
to the number of rotatable bonds.
Since effects from excluded volume increase with the number of atoms for number of rotatable bonds, the conformational entropy
ligand (ASCOnf,nP) bonds
to
is considered to be
(Nrb) and the total number of
change
for
a
given
non-peptide
a
linear function of the number of rotatable
a
(Nat):
atoms
(46)
ASml.v=k,X„+k2Nal
To date, the
applicability
tested for the
of
equation (44)
has not been
and it is
widely used,
only
analysis of HIV-1 protease inhibitors (Luque & Freire, 1998). However,
for these interactions ^
was
calK"1mor1,
found to be -1.76
whereas k2
equals
0.414
calK"'mor1.
1.9.6.
Linkage Effects
Above calculations a
second
ligand
equilibrium
is
apply for systems coupled
to
that do not involve linked
binding
of
a
linkage
interaction processes. Its contributions to the a
global analysis
If
binding of
first one, the contributions of the second
must be considered. Protonation
experimentally, using
equilibria.
is
a
common
binding energetics
of experimental data
as
phenomenon
can a
in
be determined
function of
pH,
temperature and buffer ionization enthalpy (Baker & Murphy, 1996; Baker & Murphy,
1997).
1.10. The the
Thermodynamics
and Rational
Drug Design
rapidly increasing availability of high-resolution protein
possibility
to use structural information in the
problem
of
dictating
the
accurate
prediction of
structure-based
design
studies
energetics of the interaction of
is a
design of
the
ligand
structures has new
drugs.
understanding with
a
of
opened
The central the
features
macromolecule, i.e. the
the Gibbs free energy that determines the
binding affinity.
40
PART 1: Isothermal Titration
1.10.1. The The
Thermodynamic Approach
prediction
of
binding energetics
is
greatly complicated by
entropy compensation (Dunitz, 1995; Gilli
a
conformations
Additional
in
energetic effects
free
the
The
ligands
solution
contributing
forces
no
been
is
the
to
adapting
the
predict
ligand
macromolecule.
binding pocket
is that it has become
to
possible
to
(protein-, ligand,
found in the system
not
in Table
possible
The situation area
3), based
on
the
an
now
& Makhatadze, 1993;
(Baker
has
important role in molecular
changed
(AASA)
are
of
set
to
predict the
&
Spolar
et
and
thermodynamic
the
realization that
with
related to the
structural
energetics
energetic parameters
et
of
to
The
in
drug design.
folding
of
a
globular protein
al., 1992; Murphy & Freire, 1992; Privalov
al., 1992; Xie & Freire, 1994) with
Murphy, 1998).
thermodynamic
parameterization of AG, AH, AS, and ACp
promising thermodynamic approach
a
(Makhatadze & Privalov, 1993; Murphy
which accurate
played
theoretical framework that relates structural
established.
AASA, is
9% to 12%
enthalpic
Thermodynamic Approach
in solvent-accessible surface
(summarized
It
bound
role in
are
parameters. The semi-empirically derived
terms of
and
interactions).
since
changes
entropie
(AH, AS, ACp) which make up the free binding energy
recently, thermodynamic data have
data has
improvement is only achieved
by direct calorimetric measurements, yielding effective energetic
1.10.2. Current Status of the
design,
and
important
an
contributions, including all interactions that
Until
increase
will arise from any differences in
thermodynamic approach
the
of the interaction
and solvent
an
.
strength of
dissect the
in
state
Moreover, water molecules play different
that
means
enthalpy-
to the observed AG makes it difficult to rationalize and
contributing
binding affinities.
the
as
the effects of
cost in the TAS term. The sheer number of
compensation
effects
ai, 1994) which
et
binding affinity,
in AH does not contribute to the
by
Calorimetry
parameterization
predictions of binding energetics
is
an
accuracy within
has reached the state in
possible
as
well,
as
shown for
peptide-protein and protein-protein association (Baker & Murphy, 1997; Burrows
et
al., 1994; Gomez & Freire, 1995; Murphy et al., 1993), and also for nonpeptide
ligand-protein
approach
interactions
is still in its
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PART 1: Isothermal Titration
the
design
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a
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a
Calorimetry
41
"thermodynamic-directed
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drug-
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1.10.3. Outlook The structural
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of
development
parameterization is
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and
binding,
to
still based
i.e.
and
for
set of
drug
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characterization
ACp,
but also
of
tools for the
However,
current
thorough investigation
more
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of
phenomena
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and the set of
allow accurate
new
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expanded
promising
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on
structural
only AH, AS,
not
strategies
a
prediction
energetic parameters
can
of effects of small molecular
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thermodynamic
be refined to the
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binding
constants in aqueous solution: studies
vancomycin group antibiotics. Proc. Natl. Acad. Sei. USA
1172-8.
U.
(1974). Zur Geschichte der Entwicklung der physikalischen Chemie
(Dissertation, Ed.),
Frankfurt
am
Main.
50
PART 1: Isothermal Titration
Calorimetry
Wiseman, T., Williston, S., Brandts, J. F. & Lin, L. N. (1989). Rapid
binding
constants and heats of
Biochem.
Wymann,
J.
179(1),
& Gill,
binding using
a new
measurement of
titration calorimeter. Anal.
131-7.
S. J.
(1990). Binding
Biological Macromoiecules, University
and
Linkage: Functional Chemistry of
Science Books, Mill Valley, CA.
Xavier, K. A., Shick, K. A., Smith-Gil, S. J. & Willson, R. C. (1997). Involvement of water
molecules in the association of monoclonal
bobwhite
quail lysozyme. Biophys.
J.
73(4),
antibody HyHEL-5 with
2116-25.
Xie, D. & Freire, E. (1994). Molecular basis of cooperativity in protein folding. V.
Thermodynamic
and
structural
denatured states. Proteins
19(4),
conditions 291-301.
for
the
stabilization
of
compact
PART 1:
Herpes
Herpes Virus Type
2.1.
Type
Virus
1
1
Thymidine Kinase
51
Thymidine Kinase
Herpesviruses
Herpes viruses
belong
the
to
subfamilies.
The
(HSV1
HSV2), varicella
and
betaherpesviridae entails Eppstein-Barr virus viruses
is
the
herpes
,
virus, bovine herpes virus,
zoster
Pseudorabies virus and feline
cytomegalovirus,
and the
divided
in
DNA viruses,
three
1
and 2
marmoset
herpes
herpes
virus.
gammaherpesviridae
virus saimiri and Marek's disease
coated, species-specific
are
which
alphaherpesviridae include herpes simplex virus type
equine herpes virus,
virus,
family Herpesviridae
herpes
carrying highly
The
includes
virus.
Herpes
evolved
and
extremely specialized genes exclusively coding for virus specific proteins. Infections with
herpes viruses
and humans, which
2.1.1.
are
95% of the world
cause severe
requires effective
Herpes Simplex
Herpes viruses
can
antiviral
therapy.
Virus Infections
endemic in most animals, and it has been estimated that 60 to
population is infected by members
especially by HSV1 (WHO, 1985). Typically, childhood
by
way
of
gingivastomatitis. The enters
the
peripheral
epithelial
surfaces
virus enters the nervous
trigeminal ganglion, where
nerve
the first
in
the
the viral
latency
to other
nerve
and
encephalitis.
to
not
in as
tissues and
neurons
(Roizman
viruses, HSV1 does
occurs
manifesting
cavity,
is established
variety of diseases like the
genital skin lesions, blindness
infection
HSV1
oral
herpes family,
endings innervating these
into the host DNA, but stays dormant in the causes a
of the human
system by retrograde transport
1985; Spruance, 1995). In contrast
reactivation it
and recurrent diseases in animals
&
in the
Batterson,
integrate
its DNA
cell until it is reactivated. On
common
fever blisters, but also
52
PART 1:
2.1.2. HSV1
Herpes
viruses encode
In
contrast, for virus
dispensable
a
of
variety
proteins
the
viral
inactive TK
involved
in cell culture.
with
acyclovir,
recurrences
More were
of
recently,
a
et
latency.
(TK" mutations) severely impaired (Coen
et
ai, 1989a; Coen
ai, 1996; Jacobson
of the
are
crucial role of
deletions
leading
to
ganglionic replication
acute
able to suppress the reactivation
ai, 1995; Leib
et
ai, 1990). Treatment
analog, significantly & Thin,
latency (Horsburgh that
as
a
be
to
ai, 1989b; Efstathiou et ai,
et
publication reported mutations resulting
genetic background
a
et
HSV TK specific nucleoside
still able to reactivate from
consists of
of viral DNA,
replication
Mutations such
genital and labial herpes (de Ruiter a
the
Early reports describe
1989; Jacobson et ai, 1993), and TK inhibitors
(Gebhardt
on
thymidine kinases (TK) have been shown
replication
and reactivation in mice
in mice
Thymidine Kinase
polymerase is essential for virus replication (Challberg & Kelly,
TK in reactivation of the virus from
complete
1
Latency
of which the viral DNA
1989).
Herpes Virus Type
reduces the rate of
1994).
in
truly TK" virus strains that
ai, 1998). It
et
seems
that HSV1
permits reactivation from latency independent
thymidine kinase pathway, yet the underlying mechanism remains
to
be
making
the
discovered.
However, cells of the neuronal system contain little formation of dTMP the rate
constructing
HSV either
limiting step
encoding
for their
or no
for virus reactivation. This own
and for cellular TK. Cellular TK
for reactivation,
to
phenotype Taken
partially competent viruses,
which failed to reactivate from
together, TK appears
was
shown
by
TK, for thymidylate kinase (TmpK), for
deoxycytidine kinase (dCK) TmpK led
cellular TK,
latency (Chen
et
was
found to be sufficient
and dCK exhibited
a
TK"
ai, 1998).
still to be essential for reactivation from
latency,
but the
precise mechanism remains unclear.
2.2.
Thymidine
Thymidine all
kinases
Kinases
(EC 2.7.1.21)
organisms. They catalyze
monophosphate (dTMP) phosphate
product
is
the
highly
conserved enzymes that
phosphorylation
in presence of
group of adenosine
systematically
are
magnesium
triphosphate (ATP)
correct name for TK is
of
occur
thymidine (dT) cations
to
in
nearly
thymidine
by transferring
the y~
to the 5"-OH group of dT. The
ATP-thymidine-5'-phospho-transferase.
subsequently rephosphorylated
by
cellular
thymidylate
kinase
The and
PART 1:
nucleoside
diphosphate
Herpes Virus Type
kinases to
finally
1
build the
53
Kinase
Thymidine
triphosphate (dTTP)
used for DNA
synthesis. The cell does not
synthesis
novo
depend pathway
in presence of
(dUMP)
TK is therefore
designated
This type of enzyme is
to form
dTMP, since it is provided by the deof
methylation
as a
salvage pathway
decomposition
deoxyuridine
monophosphate
recycling endogenous
enzyme,
resorbed from nutrition.
or
tissues, especially in the liver. Therefore,
in fetal
fetal TK. Prior to birth the concentration declines
or
certain
dT
Kinase
mainly found
adults, high concentrations
patients suffering
activity
through
Cytosolic Thymidine
it is also termed TK1
TK
methylene tetrahydrofolate by thymidylate synthetase (Fig.1).
released from metabolic
2.2.1.
on
only
are
found in
dividing
neoplastic diseases,
cells
sharply. In
(Kit, 1976), in the
and concomitant with
a
of
serum
number of viral
infections. TK
activity
levels
are
tightly regulated by
position
of the cell, with
low
undetectable
or
dividing
activities
both the
revealing high
cells
growth
state and the cell
activities and
resting
cells
cycle
having
(Machovich & Greengard, 1972). In mitogenically
stimulated cells, TK
activity remains low in the Gi phase, increases dramatically
the cell enters the S
phase,
and remains elevated
during S
and G2
as
phase (Carozza
&
Conrad, 1994; Kauffman & Kelly, 1991; Sherley & Kelly, 1988).
2.2.2. Mitochondrial
Thymidine Kinase
This type of TK, also named TK2, is localized in the mitochondrial matrix and exhibits
low but constant levels of TK
the
activity during
cytosol and subsequently translocated,
peptide
that is cleaved off after
involved
in
growth,
cell
but
having is
mitochondrial DNA, since dTMP is
the entire cell most
cycle.
It is transcribed in
probably guided by
reached the mitochondrial matrix for
used
thymidine
negatively charged
nucleotide
and exhibits
a
.
signaling
TK2 is not
synthesis
extremely
of
poor
penetration of the mitochondrial membrane. TK2
is able to
(FAIU),
and
it
phosphorylate dC, dU, AZT and fluoroarabinofuranosyl-iodouracil is
therefore
thought
to
be
responsible for mitochondrial toxicity
(Johansson & Karlsson, 1997; Lewis & Dalakas, 1995).
54
PART 1:
Herpes Virus Type
"salvage pathway"
1
Thymidine Kinase
"de
novo
synthesis"
O
0
HN"
CH3
HN'
O^N' k K/^0-PQ3
O^N'
\T0H
OH
2'-deoxyuridine-5'-monophosphate (dUMP)
OH
thymidine (dT)
ATP
thymidylate synthetase
thymidine \"-—*-ADP
5,10-methylenetetrahydrofolate \
kinase
I dihydrofolate-
7,8-dihydrofolate 0
CH3
HN
O-PO3
thymidine-5'-monophosphate (dTMP) ATP
thymidylate kinase ADP
dTDP ATP
nucleoside
diphosphate kinase
ADP dTTP
incorporated
Fig.1: Biosynthesis pathways
of
in DNA
by
DNA
thymidine triphosphate
polymerase
J
reductase
PART 1:
Herpes Virus Type
2.3. Structure and Function of HSV1 2.3.1.
Crystal
HSV1 TK is
consisting helices
of
Thymidine
1
Kinase
55
Thymidine kinase
Structure of HSV1 TK homodimer with 376 residues per subunit and
a
central five stranded
a
(Fig.2).
The
crystal
structure
parallel ß-sheet flanked
(Wild
ai, 1995; Wild
et
is
on
et
an
either side
residues 34-45, 150-152 and 265-279
because of insufficient electron
density
date, there
analogs
are
structures known in
and cofactor
(Bennett
ef
due to
probably
complex
by
a-
ai, 1997) comprises
residues 34 to 376, whereas
most
a/ß protein
are
missing
segmental mobility.
To
with the natural substrates, substrate
ai, 1999; Brown
et
ai, 1995; Champness
er
ai,
1998; Wild et ai, 1995; Wild et ai, 1997). HSV1 TK shows the classical mononucleotide the Walker A-motif
loop (Saraste HSV1 TK
as
ef
(Walker
belonging
to the same structural
adenylate
kinase
family
as
the NMP-kinases for which
(ADK) (Dreusicke
et
ai, 1988; Schlauderer
1996), guanylate kinase (Stehle & Schulz, 1990), uridylate kinase (Muller-
Dieckmann & Schulz, ef
ai, 1982) forming the characteristic phosphate-binding
ai, 1990). The precise ordering of the central five ß-sheets classifies
structures are known for
& Schulz,
et
(NMP) binding fold (Schulz, 1992) with
1994)
and
bacteriophage
T4
deoxynucleotide
kinase
(Vonrhein
ai, 1995). For homology analysis of NMP-kinases the central five ß-sheets has
been dubbed CORE domain whereas the domain and NMPbind domain
(Vonrhein
et
of HSV1 TK and ADK reveals substantial
Major differences formed
by
occur
kinases. Taken et
domains
are
the so-called LID
al., 1995). Superposition of the backbones
similarity
topology of
the
with respect to the CORE domain.
nucleoside/NMPbinCi domain, which is
extensive insertions, and in the size of the LID domain. The LID domain of
HSV1 TK consists of
(Wild
in the
remaining
only eight residues reminiscent of small variants of NMP-
together,
the
largest differences
are
due to the terminal residues
al., 1997). Interestingly, many of these differences appear to be connected to
the dimer interface that is similar to the dimer interface of the dimeric
kinase
(Lavie
et
deoxynucleotide
thymidylate
ai, 1997), but differs significantly of the dimeric bacteriophage T4 kinase
(Teplyakov
et
ai, 1996),
PART 1:
56
Fig.2: Ribbon diagram of the X-ray are displayed as spaced filled
2.3.2.
Herpes
Virus
structure of the
Type
1
Thymidine Kinase
symmetric HSV1
TK dimer. Bound ADP and dTMP
Quaternary Structure
In contrast to TK1, which appears to be active
as a
1993),
HSV1 TK is found to be dimeric in solution
crystal
state
(Wild
et
(Wild
et
comparable molecular weight, T4
a
Ä2
in
ai, 1994) and in the area
per subunit, i.e. 14% of the total subunit
agreement with values from
but differs
ai,
grossly
other
non-
of
dimers
from the small interface of 900
Â2
of
deoxynucleotide kinase.
The interface is almost
anchoring in
ef
al., 1997), and is, with 65% hydrophobic residues, exceptionally
polar. The large interface is
bacteriophage
(Fetzer
et
al., 1995; Wild et al., 1997). The solvent accessible surface
buried in the HSV1 TK dimer is 1800 surface
(Munch-Petersen
tetramer
planar with only
one
substantial
protrusion formed by W310
hollow of the other subunit. The dimer interface of HSV1 TK consists
of
mainly hydrophobic helix/helix interactions
is
completed by helix o2, parts of loop oc2-a3, loop a12-a13 and
of helix a15. Most of these elements
are
with oc4 and cc6
forming
the center and
the C-terminal end
part of the NMPbind domain, but there
also contributions from the CORE domain and from the additional mobile 72 residues to the interface.
are
segment of
PART 1
2.3.3.
The
Thymidine Binding
substrate
binding
thymidylate interacting dT
is
is
or
located
dTMP
kinase function of TK
via
the
in
between M128 and
by
Thymidine Kinase
so
57
(Chen
ef
a
the
additional
ai, 1979a). The carboxamide of Q125 is
thymine ring of
two water-mediated H-bonds.
Y172, forming
NMPbinci domain which
called
(Fig.3), being responsible for
H-bonds with N3 and 04 of the
linked to R176
1
Site
site
either dT
accommodates
Herpes Virus Type
sandwich-like
the substrate, the 02 of
The
complex.
thymine ring The nbose
is
held
moiety
is
interacting with Y101 and E225 The
nucleoside/nucleotide
binding
site
is
deeply
buried
in
agreement with the low KM value of 0.2 uM for dT (Fetzer Gerber et ai,
completely fill
1999; Michael ef al, 1995) its
binding pocket
It leaves
a
35
the protein
ef
in
ai, 1993; Kussmann-
Although tightly bound, Â3
interior,
dT does not
void close to its C5 position.
58
PART 1:
Herpes Virus Type
part of the ATP binding site is the glycine rich motif connecting ß-strand ß1
A crucial
fingerprint 56GXXGXGKT63 (P-loop (Saraste
with helix a1. It contains the sequence
al., 1990)), forming group of ATP in
binding
number of
a
site is much as
R220, and R222
are
kinases
by
Mg2+ together
1998).
As
deduced &
2.3.5. Substrate
Specificity
exhibits
thymidylate
is
(Walker
et
the
stereochemical
demands,
phosphorylates
binding
D-dT. The
TK is
HSV1 TK
Moreover, since
it
motif,
to take
part in
(Kussmann-Gerber
side chains
as
preferred phosphate
as
a
ef
ai,
multifunctional enzyme and
deoxycytidine kinase and
spectrum of pyrimidine
displays
low
modified
accepts
cytidine triphosphate (CTP),
well
as
binding pathway
kinases follow
a
well
stereoselective
ribose
moieties
as
and and
the L-dT instead of the natural
donor is ATP, but HSV1 TK shows
uridine
triphosphate (UTP)
is not yet clear. It is
random bi bi mechanism
and
high
guanosine
(Roads
generally accepted that NMP
& Lowenstein, 1968; Yan & Tsai,
1999). Kinetic analysis of HSV1 TK revealed different results. On the random bi-bi mechanism is
proposed (Chen
preferred, sequential binding 1999).
as
well.
The substrate
Gerber et al.,
site includes
al., 1982). It is highly conserved among
It shows additional
diversity.
acyclic
even
triphosphate (GTP)
loop, residues R212,
ai, 1995), and D162 is assumed
ef
H-bond
one
Mg2+-NTP binding
so-called
kinase activities and converts a broad
(Fig.5).
affinities for
which
rich
The bottom of the
highly specific TK1, HSV1
purine analogs
occurring
binding.
glycine
al.,
et
Schulz, 1992).
broad substrate
a
jiM (Chen
ADK, K62 is essential for phosphoryl transfer during
catalysis (Muller
In contrast to the
value of 16 to 70
high Km
with T63 and three water molecules
from
y-phosphoryl
exposed (Fig.4), and the adenosine moiety is
R216. Besides the
Walker
(Remond
binding
and
al., 1999). The adenine moiety is bound by
FD1S2RH,
originally identified by thymidine
et
involved in ATP
motif
sequence
solvent
reflected in the
and flanked
ß-
et
ATP-binding proteins (Schulz, 1992).
more
1979a; Kussmann-Gerber
through Q331
anion hole that accommodates the
giant
a
only weakly bound
the
Thymidine Kinase
Binding Site
2.3.4. ATP
The
1
et
ai, 1979b), and
order has been described
From the structural
point
on
hand
a
the other hand
a
(Gerber, 1997;
one
Kussmann-
of view it would be assumed that dT
PART 1
Fig
4-
Representation
entry 1VTK) sticks,
Herpes Virus Type
of the nucleoside/nucleotide
Amino acids
hydrogen-bonds
are
enzyme. The LID domain
directly involved displayed
as
well
as
as
the
in
Thymidine
1
binding site
cofactor
Kinase
59
of HSV1 TK with bound dT
binding
labeled and shown as
are
dashed lines Tubes represent the
glycine-nch loop (P-loop)
(from
are
secondary
indicated. The
PDB
capped
structure of the
figure
has been
prepared using SYBYL V 6.3 (Tripos Associates)
must bind
ATP
first, since its binding
placed in front like
studies and
thermodynamic
2.3.6. Structural
Catalysis by that
occur
transfer
a
site is localized
plug. This view
due to
kinases is known to be
during
substrate
phosphoryl groups
to
is further corroborated
by induced-fit
and cofactor
hydroxyl
binding. Since
groups,
they need
movements that exclude
crystallographic analysis
with
by mutagenesis
accompanied by large conformational changes
from the reaction center of the kinases, detailed
protein,
Binding
from the surrounding water to avoid ATP hydrolysis achieved
in the
(this work).
measurements
Rearrangement
deeply buried
as
it
to
in
most
of conformational
kinases
protect their active sites
(ATPase function).
strongly competing is
cases
water
This
is
efficiently
observed for several kinases. A
changes
of ADK showed
a
rigid
60
PART 1
Herpes
Virus
Type
1
Thymidine
Kinase
0
0
iL
II
TlN^Ti
\_T\m
vT
\
on
OH
2'-deoxyuridine
thymidine 0
Nil, "
1
(Y
N
on
N
\_r
vT
\m
ou
on
OH
2'-deoxycytidine
idoxuridine
NIT
0
N
> N N
1 UN
7
V V\,r
K
\-,H
vidara bine
acyclovir
OH
/
0
A
[ y
n2N-^N^
n
Y""^oft penciclovir
Oil
N
H2N
^N
/^-OII
^on
lobucavir 0
0
I
HN
jr3
CT
Y^^OH Oil
ganciclovir Fig.5: Formula of various HSV1 TK substrates and analogs thereof
if N
\ AZT
/
on
4\> N~
PART 1.
Herpes Virus Type
Fig.6: Ribbon diagram of adenylate kinase AP5A (PDB entry 1AKE),
inhibitor
from E.coli.
1
Thymidine Kinase
(A) ADKeco
which mimics both substrates.
complex with the dinucleotide
in
(B)
The
(apo) stage (PDB entry 4AKE). The domains NMPbl„d (30 residues) relative to the two helices
domain
packed together
four localized
responsible
rigid CORE
(138 residues)
in an
regions, called joints,
for
approximately
antiparallel
The LID domain is linked to the rest of the
During
fashion.
60
degrees. These
movements carries
for
the
a
first
along
domain
accompanied by large hinge bending
rotation
to the NMP
kinases, structures
ATP, either separated To date, there is a
mutated
no
or
in form of
a
ADK in
substrates,
are
complex
with
an
solved.
pair of joints is
inhibitors
and
as
between the
rigid
a
the two
bodies.
LID
domain
(Fig.6) (Muller
known for
joints,
et
complexes
movement
ai, 1996). With
with NMP
or
with
ATP
alone, with the only exception
derivative
(ADPCF2P), showing
are
et
ai, 1996).
ternary complexes with natural and
cofactors
or
sulfate
the
ions
(mimicking
non-
the
ß-
to the closed conformation of NMP kinases
al., 1995), whereas structural data corresponding to the native apo form
has not yet been
binding
and
regions
binding (Schlauderer
phosphate of ATP), i.e. they correspond et
the
structure known with natural ATP
All structures of HSV1 TK solved to date
(Vonrhein
place in
covalent connection of two nucleotides.
conformational changes related to ATP
natural
move
protein by
the closure, deformations take
approximation,
NMP-binding
of
(38 residues)
LID
and
enzyme in the free
approximately 30 degrees of the total rotation and the second pair for the remaining
rotation
respect
same
the N- and C-termini of these helices. The first
near
helices and the rest of the mobile domain, to
body
61
reported. Therefore, the
of HSV1 TK with respect to
exact mechanism of substrate and cofactor
possible
structural rearrangement remains to be
62
PART 1:
Chemotherapy and the
is
Drug Target
as
of viral infections is difficult, since viruses possess
is based
elevated
enzymes which
selectivity
infected cells.
essential for
are
First treatment of introduction of
in
widely
only given by
Several
DNA viruses
acyclovir, and HSV1
Both virustatics
are
are
virus
the
1978), further phosphorylated
synthesis
encode for their
rate own
started at the end of the 1970's with the
TK was
soon
identified
by HSV1 TK to their
as
the target enzyme for
therapeutic prodrugs (Fyfe
purine analogs acyclovir (ACV)
activated
the DNA
are
replication, i.e. TK and DNA polymerase.
herpesvirus infections
used
metabolism,
analogs. However,
is
the activation of selective and effective most
no
host cell enzymes, thus infected and non-infected cells
on
A certain virus
equally damaged. which
Thymidine Kinase
of antimetabolites is limited to nucleoside
use
replication
1
HSV1 TK
Aspects of
2.4. Medicinal 2.4.1. HSV1 TK
Herpes Virus Type
to their
and
et
ai, 1978). The
gancyclovir (GCV).
monophosphates (Fyfe
triphosphates by
cellular enzymes
et
al.,
(Miller
&
Miller, 1980) and incorporated into DNA, resulting in chain termination and formation of dead-end
difference
complexes
in
substrate
with viral DNA
specificity
between
molecular basis for this selective antiviral
Although
TK is not
pathogenicity
TK1
and
a
inhibit HSV1 TK
generally required for
number of groups have
(Gebhardt
et
Spector, 1989).
HSV1 TK
establishes
The
the
therapy. virus
replication,
in animal models and for reactivation from
Consequently,
&
polymerase (Reardon
required for full
it is
latency (Coen
synthesized compounds
et
al., 1989b).
that
specifically
al., 1996; Hildebrand et ai, 1990; Martin et ai, 1989;
Martin et ai, 1998; Martin et ai,
1983), without being phosphorylated, and which
are
able to diminish reactivation of viruses. Since non-neuronal tissues exhibit cellular TK
activity, specific viral TK inhibitors will have little
(Gebhardt
et
prophylaxis
al., 1996;
synthesis.
latency
no
1990). This class of
of infection and reactivation of
virus is reactivated from
viral DNA
Leib et al.,
or
latency,
effect
on
the
host cell
antivirals acts towards
but will be ineffective
because non-neuronal tissues will
provide
once
the
dTMP for
Herpes Virus Type
PART 1:
2.4.2. HSV1 TK and Gene
foreign
feasible to insert
genes into
new
based
63
Thymidine Kinase
Therapy it became
development of biotechnological methods for gene manipulation,
With the
of
1
introducing
on
susceptible features
a
to
genes into viral
metabolic property into
a new
a
that will be activated
unique properties which
are
therapy
of suicide gene
general concept
human cell. The
therapeutic drugs,
some
bacterial gene vectors, and also transfer
or
is
to make it
target cell in order
in these cells. HSV1 TK
only
exploited for several different gene
therapeutical approaches. the
For
of
treatment
neoplastic
developed (Huber
enzyme/prodrug therapy has
been
transduces the tk gene into
population
a
drug.
The
a
of cells,
virus-directed
so-called
the
diseases,
ai, 1991). This approach
et
conferring
lethal
a
sensitivity
to the
major problem of this approach is the specific targeting of malignant
cells, selective gene delivery, specific gene expression, specific gene activity and, if
possible, specific drug
challenge (Dachs
et
To date,
number of
types
cell
lung
cancer
(Kumagai
ai, 1997; Tong
gastrointestinal Further
cancer
ef
tumors
(Yang
(Charissoux
the field of AIDS
cancer
(Tong
ai, 1999; Tong
et
et
ai, 1998), uterine adenomcarcinoma (Kunishige et ai, 1999), ef
ai, 1998), and colon
cancer
be treatment of retinoblastoma
applications might
osteosarcoma
a
under evaluation for this kind of treatment, i.e.
are
ai, 1996), ovarian
ef
remain
problems
ai, 1997).
formidable a
recent progress, these
Although
action.
et
ai, 1999),
or
pituitary
tumors
(Wildner
(Hurwitz
(Lee
ef
et
ef
ai, 1999).
ai, 1999),
ai, 1999). Even in
therapy, the gene therapy approach has been adopted (Caruso &
Klatzmann, 1994; Christians et ai, 1999; Guettari et ai, 1997).
Phosphorylation of New
2.4.3.
substrate
exceptional
The
phosphorylation of such
compounds
non
DNA
Building Blocks
diversity
of
HSV1 TK
standard nucleosides. Since
is the rate
limiting step,
TK
can
DNA
building blocks,
HSV1 TK may be active
thus
engineered
(Pilger, 1999).
extending
to allow
the
forming
be used
phosphorylation step. Subsequent phosphorylation new
can
to the
the
exploited
the
monophosphate
as a
of
device to for the first
triphosphate
genetic alphabet.
phosphorylation,
for
be
would lead to
For such purpose,
if wild type enzyme is not
PART 1:
64
Herpes
Virus
Type
1
Thymidine Kinase
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ff
\;ol
PART 1:
Periplasmic
Periplasmic Histidine Autokinase CitA
73
Histidine Autokinase CitA
3.1. Citrate Metabolism Citrate is not
an
ubiquitous
surprising
source.
natural
compound that
that many bacteria
are
occurs
in all
living cells.
able to utilize citrate
as
It is therefore
carbon and energy
Under aerobic conditions citrate is usually metabolized via the
tricarboxylic
cycle (TCA cycle), Citrate fermentation under oxic conditions requires only
acid
additional citrate carrier for citrate uptake and, if citrate is the enzymes that allow formation of
acetyl-CoA
and
only substrate,
a set
phosphoenol-pyruvate from
an
of
TCA
cycle intermediates. Anaerobic conditions TCA
cycle
is
usually
requires
different
a
operative
not
due to
oxoglutarate dehydrogenase (Park
ef
pathway for
citrate dissimilation, since the
suppressed synthesis of the key enzyme 2-
ai, 1997). A number of different pathways for
citrate fermentation have been discovered do date (Antranikian & Giffhorn, 1987), of which
the
cleavage
only
enzyme
of citrate
into
in
is citrate
common
acetate
and
lyase.
oxalacetate.
indicative for anaerobic citrate fermentation
This
enzyme catalyzes the
Consequently,
its
presence
is
capabilities.
3.1.2. Citrate Fermentation in Klebsiella Pneumoniae
Klebsiella pneumoniae utilize citrate
as
prerequisite for are
known
belongs
to the
family
the sole carbon and energy
citrate fermentation is its
of Enterobacteriaceae and is able to source
under anaerobic conditions. A
uptake by carriers, of which
at least three
(Schwarz & Oesterhelt, 1985). Two have been analyzed genetically and
biochemically.
During
aerobic
independent
growth,
and is driven
citrate
uptake
mainly by
a
by
the
CitH
carrier
transmembrane chemical
protein
is
sodium
gradient of H+
and
74
Periplasmic Histidine Autokinase CitA
PARTI
H*2Na+
Fig.1: Schematic
view of citrate fermentation
pathway
in
Klebsiella
pneumoniae.
For details see text
(from Meyer, 1999).
by
a
be the
(Van
transported species, and
The divalent anion
uptake
across
the
has been shown to be
cytoplasmic
H-citrate2"
is assumed to
symport with three protons has been proposed
a
der Rest et ai, 1991; Van der Rest et ai,
citrate
H+
gradient.
transmembrane electrical
dependent
1990). Under fermentative growth,
on a
chemical
gradient
membrane. The process is mediated
by
of Na+ and/or
the dimeric CitS
carrier protein (Pos ef ai, 1994; Pos & Dimroth, 1996; Van der Rest ef ai, 1992). The first step of citrate acetate
and
degradation
oxalacetate
(Fig.1).
membrane bound oxalacetate
which generates
an
of citrate is
Latter
is
formate
lyase
acetyl-phosphate.
to
The
by substrate level phosphorylation
oxalacetate ATP
during
citrate to
lyase
to form
by
pyruvate
the
decarboxylase Na+ pump (Bott, 1997; Dimroth, 1988),
gradient. Pyruvate
yield acetyl-CoA, which is converted
phosphate by phosphotransacetylase. Finally,
per mol of citrate
decarboxylated
transmembrane electrochemical Na"
degraded by pyruvate
from ADP and
cleavage by
is further to
acetyl-
acetate kinase is used to form ATP
pathway allows the synthesis of
1 mol of ATP
in the acetate kinase reaction, and 0.3 mol ATP
by converting the electrochemical Na+ gradient established by
decarboxylase (Bott, 1997). fermentative growth
on
These are the
only
reactions that
citrate under anaerobic conditions.
provide
PART 1:
Regulation
3.2.
Periplasmic Histidine Autokinase CitA
of Citrate Fermentation
The environmental conditions of
monitor their
surroundings
intracellular
by
microorganisms
either
directly by
cascade
initiated
is
and
leads
activities, chemotactic behavior, and i.e.
found,
been
the
to these
altered
to
a
Chemotaxis
changes.
Bacteria
for
indirectly
sensors
metabolite
stimulus is detected,
transducing systems
a
the
and
have
phosphoenol-
the
proteins,
systems,
rapid changes,
expression, enzymatic
gene
A number of
to
cytoplasmic
the
in
changes. Once
phosphotransferase
pyruvate:carbohydrate
subject
membrane-bound
more.
methyl-accepting
often
adapt
detecting fluctuations
receptors,
concentrations that result form extracellular
signaling
are
must be able to detect and
microorganisms
and
75
two-component
& Stock, 1998; Hazelbauer et ai, 1993; Postma ef ai,
regulatory systems (Goudreau 1993).
3.2.1.
Two-Component Regulatory Systems
Two component systems
archaea and several and A
are
formed
are
eukaryotic organisms (Posas
involved in the
regulation
regulator, representing
two
the
components contains
signal
transduction
protein/protein
interaction
Goudreau & Stock, Extracellular
of
presence conserved
consists of
the
are
signal,
histidine
Information
pathway.
a sensor
transduction
kinase and
occurs
by protein phosphorylation (Appleby
and
detected the
homodimeric of
domains of the
by periplasmic
the
sensor
transmitter
240 amino acids in
signal
regulator by
length (Stock
ef
is passed onto
means
of
a
kinase
domain
in
a
ef
by
direct
ai,
1996;
the second
domain of the
kinases. In
autophosphorylates trans,
ATP
using
a as
ai, 1995). From the phosphorylated
a
conserved aspartate residue of the
reversible
phosphotransfer function (Wanner, 1995).
sensor
in the transmitter domain and is
Mg2' dependent phosphotransfer
reaction. The receiver domain is about 120 amino acids in
activates
a/., 1994)
at least two functional domains and form the backbone of
residue
transmitter domain the
response
ef
modules. Either of these
signal-communicating
phosphoryl donor. The kinase function is included
generally
a/., 1998; Swanson
1998).
changes the
ef
eubacteria,
of citrate fermentation.
prototypical two-component regulatory system
response
in
by ubiquitous proteins found
The
length
phosphorylated
regulatory protein, leading
and harbors the
receiver to the
domain
response
76
PART 1:
to the
according
Periplasmic Histidine Autokinase CitA
type of regulatory system, i.e. binding
target DNA sequence
to a
or
methylesterase activity. Inactivation is achieved either by the response regulator through its autophosphatase activity, alternative
3.2.2. In
an
are
with
proein
or
by
it
cognate
kinase,
sensor
or
noticed that the
was
to catabolite
an
of Klebsiella Pneumoniae
responsible enzymes in citrate fermentation
induced under anaerobic conditions in presence of citrate, and that
subject
by
autophosphatase activity.
Regulation of Citrate Fermentation early study
the
they
are
repression (Dagley & Dawes, 1953). Recent investigations of the
genes required for expression of the Na+ dependent CitS citrate carrier led to the identification of Klebsiella
a
two-component regulatory system essential for anaerobic growth of
pneumoniae (Bott
ai, 1995). The system consists of the
ef
sensor
kinase
CitA and the response regulator CitB. The
sensor
structure
kinase
indicated
aminoterminal
CitA is 547 amino acids in length. that
the
protein
membrane
is
periplasmic domain enclosed by
Analysis of
bound
and
the
primary
composed of
two transmembrane
helices,
a
an
central
cytoplasmic domain, and carboxyterminal cytoplasmic kinase domain harboring the
autophoshorylable histidine highly specific The
response
residue. The
citrate receptor
regulator
is
carboxyterminal output
composed of
containing
domain with
a
234
the
amino
acids
and
consists
In
region
helix-turn-helix motif
of the citS operon of Klebsiella
general, citrate fermentation
triggers
the
phosphorylation
pneumoniae (Meyer
et
to DNA
ai, 1997). Na+ which
catalyzed by the CitA/CitB two-component
system. Phosphorylized CitB activates transcription of the citS promoters, leading elevated levels of the citrate carrier
keeping enzymes
are
termination
al., 1995).
(Bott
et
controlled
an
typical for DNA binding
is achieved in presence of citrate and
cascade
of
phosphorylable aspartate residue, and
proteins. Recently, it has been shown that the carboxyterminal domain binds of the control
as a
(this work).
aminoterminal receiver domain a
periplasmic domain has been defined
to
protein CitS. Adequate levels of the house¬
by mRNA processing and partial transcription
PART 1:
Periplasmic Histidine Autokinase CitA
77
3.3. References
Antranikian, G. & Giffhorn, F. (1987). Citrate metabolism in anaerobic bacteria. FEMS Microbiol. Rev. 46, 175-198.
Appleby,
J. L,
Parkinson, J. S. & Bourret, R. B. (1996). Signal tranduction via the
multi-step phosphorelay:
not
necessarily
road less traveled. Cell 86, 845-
a
848.
Bott, M. (1997). Anaerobic citrate metabolism and its regulation in enterobacteria. Arch. Microbiol. 167, 78-88.
Bott, ML, Meyer, M. & Dimroth, P. (1995). Regulation of anaerobic citrate metabolism in Klebsiella
Dagley, S.
pneumoniae. Moi Microbiol. 18(3),
& Dawes, E. A.
(1953). Citric
533-546.
acid metabolism of Aerobacter aerogenes. J.
Bacteriol. 66, 259-265,
Dimroth,
P.
(1988).
The
role
of
vitamins
fermentation. In The Roots of Modern v.
& Jaenicke,
their
and
carrier
proteins in citrate
Biochemistry (Kleinkauf, H., Döhren,
H.
L, eds.), pp. 191-204. Walter de Gruyter and Co., Berlin.
Goudreau, P. N. & Stock, A. M. (1998). Signal transduction in bacteria: molecular mechanisms of
stimulus-response coupling.
Curr.
Opin.
Microbiol. 1, 160-169.
Hazelbauer, G. L, Berg, H. C. & Matsumura, P. (1993). Bacterial motility and signal transduction. Cell 73, 15-22.
Meyer,
M.
(1999).
Regulation
of
the
citrate
fermentation
genes
Klebsiella
in
pneumoniae: functional analyis of the two-component system CitA/CitB and of the cAMP receptor
Meyer, M., Dimroth,
P. & Bott, M.
CitB and of its
the
control
pneumoniae.
protein, Dissertation ETH. (1997).
carboxy-terminal
region
of
the
In vitro
domain to A
binding +
divergent citC
of the response
regulator
T-rich DNA target sequences in
and
citS
opérons
of
Klebsiella
J. Moi Biol. 269, 719-731.
Park, S.-J., Chao, G. & Gunsalus, R. P. (1997). Aerobic regulation of the sucABCD genes of Escherichia coli, which encode
succinyl
coenzyme
A
synthetase:
sdhCDAB promoter. J. Bacteriol.
roles
179(13),
a-ketoglutarate dehydrogenase of
ArcA,
Na+-dependent
347, 37-41.
citrate carrier of
Fnr, and the upstream
4138-4142.
Pos, K. M., Bott, M. & Dimroth, P. (1994). Purification of the
and
Klebsiella
two active fusion
proteins of
pneumoniae. FEBS Letters
78
PART 1: Periplasmic Histidine Autokinase CitA
Pos, K. M. & Dimroth, P. (1996). Functional properties of the purified Na+-dependent citrate carrier of Klebsiella pneumoniae: evidence for the carrier
protein
in
asymmetric
proteoliposomes. Biochemistry 35,
orientation of
1018-1026.
Posas, F., Takekawa, M. & Saito, H. (1998). Signal transduction by MAP kinase cascades in
Postma,
P.
budding yeast. Curr. Opin. Microbiol. 1(2), Lengeler,
W.,
J.
W.
&
175-182.
G.
Jacobson,
(1993).
R.
Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol. Rev.
Schwarz,
E.
57(3), 543-594.
Oesterhelt,
&
pneumoniae
D.
(1985).
Cloning
and
expression
of
Klebsiella
genes for citrate transport and fermentation. EMBO J.
4(6),
1599-
1603.
Stock, J. B., Surette, M. G., Levit, M. transduction
catalysis.
In
&
Park, P. (1995). Two-component signal
systems: structure-function
Two-component signal
relationships
transduction
(Hoch,
and
mechanisms
of
Silhavy,
J.,
J. A. &
T.
eds.), pp. 25-51. ASM Press, Washington, D.C. Swanson,
R.
V.,
L.
Alex,
A,
&
Simon,
M.
phosphorylation: two-component systems
I.
(1994).
Histidine
and the limits of
and
aspartate
homology.
Trends
Biochem. Sei. 19, 485-490. Van
der
Rest, M., Molenaar, D. & Konings, W. N.
dependent
citrate
(1992). Mechanism of Na+-
transport in Klebsiella pneumoniae. J. Bacteriol. 174, 4893-
4898.
Van der Rest, M. E., Abee, T., Molenaar, D. & and
energetics of
a
Konings,
W. N.
(1991).
Mechanism
citrate-transport system of Klebsiella pneumoniae.
Eur. J.
Biochem. 195, 71-77.
Van der Rest, M. E„ Schwarz, sequence of
a
E„ Oesterhelt, D. & Konings, W. N. (1990). DNA
citrate carrier of Klebsiella
pneumoniae. Eur.
J. Biochem. 189,
401-407.
Wanner, B. L. (1995). Signal transduction and
cross
regulation
in the Escherichia coli
phosphate regulon by PhoR, CreC, and acetyl phosphate. In Two-component signal transduction (Hoch, J. A. & Silhavy, Press, Washington, D.C.
T. J.,
eds.),
pp. 203-221. ASM
PART 1: Aims and
Objectives
Specific binding
is
a
critically,
depend
fundamental process
be
which
on
important
to obtain
complex formation.
virtually
macromolecule-macromolecule
it
molecule interactions. In order to understand and very
79
of the Presented Work
the Presented Work
Scope of
Aims and
4.1.
Scope
a
macromolecule-small
or
it is
manipulate these processes,
description of
close and detailed
biological systems
all
This includes the determination of
the forces that drive
changes of
all
thermodynamic
parameters, including free energy of binding (AG), enthalpy (AH) and entropy (AS) of
binding is the
and the heat
capacity change (ACp).
point of view with respect type
1
domain
et
therapy of
ef
proliferative
being used
as
important target
thermodynamic
herpes simplex
virus
of
interactions
ligand-receptor
in medicinal
therapy of
the
cancer
chemistry (Culver
et
because of its links
ai, 1994; Kunishige
ai, 1998) and AIDS (Caruso & Bank, 1997; Smith et ai, 1996), disorders
(Ohno
control system in
ef
encephalitis (Iwashina
et
ai, 1994; Xu
allogeneic
ai, 1997) and AIDS vaccine (Chakrabarti viral
of
interactions of
a
of the histidine autokinase CitA.
viral infections, gene
ai, 1999; Tong
vascular
an
and
aspect of the presented work
characteristics from
ligand-enzyme
(HSV1 TK)
(CitAP)
HSV1 TK has become
with
to the
thymidine kinase
periplasmic
binding
and elucidation of
investigation
The main
et
ai, 1988) and
ai, 1999). For above applications,
a
bone
ef
ai, 1998). In addition, it is
marrow
transplantation (Bonini
ef
ai, 1996), for non-invasive diagnosis of as
expression reporter gene (Gambhir
variety of compounds
is
required
ef
that will
comfort to the individual needs of the system different systems. The fact that these
compounds
are
not
concerning ligand
readily synthesized clearly shows the lack of knowledge
interactions
of
HSV1 TK.
A
deeper understanding
of
these
PART 1: Aims and
80
interactions from the
of
design
new
To achieve this, the
as
a
thermodynamic point
and effective
of the Presented Work
of view will contribute to the
compounds.
interactions
characterizing
for
tool
parameters. Therefore, several specific objectives As
a
protein,
the establishment of
was
of
very unstable, and it
necessary to
was
objective
had to
optimized.
The
to pure and active fusion
laboratory the expression and
established
for
optimized
and
measurements and
was
develop modified
high yield of
and
devoted
crystallization
protocols
with
development of proper experimental Conditions
experiments.
and verified in order to avoid
and refined
the macromolecule.
the
to
conditions suitable for microcalorimetric
optimized
our
been
thermodynamic
ai, 1994). However, it has been shown that the kinase is
ef
stability, quality
The second
has
protein suitable for kinetic
pure
(Bohner, 1996; Fetzer
to
HSV1 TK
recombinant
obtaining highly
respect
strategy that led
a
of
terms
purification
free of substrate and cofactor. Recently, in
purification
in
formulated.
are
first objective, the expression, isolation and
major goal
development for
applied isothermal titration calorimetry (ITC) which has emerged
we
premier
Scope
possible systematic
subsequently
were
errors
(pH-dependent
activity, kinetic behavior, CD spectroscopy, HPLC). The
thermodynamic
denoted the third
characterization and
objective. ITC
forming
of all elementary steps
exploration of ligand binding
has been used to
the
investigate
to HSV1 TK
binding energetics
the
catalytically competent state, in the temperature
range of 10-25°C. Experimentally obtained thermodynamic quantities should then be linked with structure-based the ternary TK:dT:ATP
Objective
four
mutants
of
distinguishable
complex of
based
residue
triad
on
binding energetics of several
that
H58/M128/Y172
from each other by kinetic measurements. The
binding behavior of sensor
pneumoniae, in
citrate
might
kinase of the two-component
which is
responsible
fermentation
function
to
and
background of
non-binding
or
not
this
altered
the mutants.
under
component regulatory system CitA
inactive
were
objective is whether loss in phosphorylation activity is due
CitA is the
the known structure of
HSV1 TK.
concerned with the elucidation of
was
the
thermodynamic analyses
as a
regulatory system CitAB of
for the induction of
anaerobic
occurs
conditions.
responsible The
enzymes involved
activation
of
the
two-
proposed
that
1995). The main focus of
this
in presence of citrate, and it was
citrate receptor (Bott ef ai,
K.
PART 1: Aims and
system
the
was
investigation of
Scope of
the sensory
81
the Presented Work
periplasmic
domain of
experimental
conditions
of the
properties
the histidine autokinase CitA.
Therefore, the fifth objective to
ITC.
perform
was
the
work,
this
For
the establishment of proper
receptor
recombinantely overproduced periplasmic histidine tag
(CitAPHis)-
The seventh
objective
binding
to
was
CitAPHis dependent
derivatives isocitrate and The
devoted to the
domain
available
with
was
the
C-terminally attached
a
thermodynamic characterization
pH, Specificity
of
form
in
explored by
means
of citrate
of citrate
tricarballylate.
thermodynamic approach
lot of information about
on
was
turned out to be very efficient and useful,
recognition
processes between small
ligands
comprising
and
a
biological
macromolecules.
4.2. References Bohner, T. (1996). Expression, purification, crystallization and structure determination of
herpes simplex type
1
thymidine kinase, Dissertation
ETH Zurich.
Bonini, C, Ferrari, G., Verzeletti, S., Servida, P., Zappone, E., Ruggieri, L, Ponzoni, M., Rossini, S., Mavilio, F., Traversari, C. & Bordignon, C. (1997). HSV-TK gene transfer into donor lymphocytes for control of leukemia
allogeneic graft-versus-
[see comments]. Science 276(5319), 1719-24.
Bott, M., Meyer, M. & Dimroth, P. (1995). Regulation of anaerobic citrate metabolism in Klebsiella
pneumoniae.
Moi. Microbiol.
18(3),
533-546.
Caruso, M. & Bank, A. (1997). Efficient retroviral gene transfer of
herpes simplex
52(2),
virus
thymidine
kinase gene for HIV gene
a
Tat-regulated
therapy.
Virus Res.
133-43.
Chakrabarti, B. K., Maitra, R. K„ Ma, X. Z. & Kestler, H. W. (1996).
A candidate live
inactivatable attenuated vaccine for AIDS. Proc. Natl. Acad. Sei. USA
93(18),
9810-5.
Culver, K. W., Van Gilder, J., Link, C. J., Carlstrom, T., Buroker, T., Yuh, W., Koch, K., Schabold, K., Doombas, S. & Wetjen, B. (1994). Gene therapy for the treatment of
malignant
brain tumors with in vivo tumor transduction with the
herpes simplex thymidine 5(3),
343-79.
kinase
gene/ganciclovir system.
Hum. Gene Ther.
PART 1: Aims and
82
Scope
of the Presented Work
Fetzer, J., Michael, M., Bohner, T., Hofbauer, R. & Folkers, G. (1994). A fast method for
pure recombinant
obtaining highly
kinase. Protein
Expr. Purif. 5(5),
herpes simplex
virus type 1
thymidine
432-41.
Gambhir, S. S., Barrio, J. R., Phelps, M. E., Iyer, M., Namavari, M., Satyamurthy, N.,
Wu, L., Green, L A., Bauer, E., MacLaren, D. C, Nguyen, K., Berk, A. J.,
Cherry, gene
S. R. & Herschman, H. R.
expression
in
living
Natl. Acad. Sei. USA
(1999). Imaging adenoviral-directed reporter
animals with
96(5),
positron
Proc.
tomography.
emission
2333-8.
Iwashina, T., Tovell, D. R., Xu, L, Tyrrell, D. L, Knaus, E. E. & Wiebe, L I. (1988).
Synthesis
and antiviral
diagnosis
of
activity of IVFRU,
a
for the non-invasive
potential probe Des. Deliv.
herpes simplex encephalitis. Drug
3(4),
309-21.
Kunishige, I., Samejima, Y., Shiki, Y., Moriyama, A., Meruelo, D., Saji, Y.
(1999). Suicide gene therapy
herpes simplex
virus
F. & Murata,
for human uterine adenocarcinoma cells
using
thymidine kinase. Gynecol. Oncol. 72(1), 16-25.
Ohno, T., Gordon, D., San, H., Pompili, V. J., Imperiale, M. J., Nabel, G. J. & Nabel, E. G.
(1994).
arterial
Gene
therapy
for vascular smooth muscle cell
injury [see comments]. Science 265(5173),
Smith, S. M., Markham, R. B. & Jeang,
immunodeficiency
virus type 1
K. T.
X. W.,
Engehausen,
D.
(1996). Conditional reduction of human
replication by
G., Kaufman,
C. T., Oehler, M. K., Kim, T. E.,
R. H.,
a
gain-of-herpes simplex
ganciclovir
Anticancer Res.
ovarian
in adenovirus mediated
18(2A),
virus 1
7955-60.
Agoulnik, L, Contant, C, Freund,
Hasenburg, A., Woo, S.
(1998). Improvement of gene therapy for instead of
after
781-4.
thymidine kinase function. Proc. Natl. Acad. Sei. USA 93(15), Tong,
proliferation
L & Kieback, D. G.
cancer
by using acyclovir
thymidine kinase gene therapy.
713-8.
Xu, L F„ Xu, D. H„ Ge, K., Zheng, Z. C, Sun, L. Y. & Liu, X. Y. (1998). The suicide gene systems in vascular cells in vitro. Cell Res.
8(1),
73-8.
use
of
PART 2
PART 2
Experimentals
"*
\
\
*
VI>
^^
PART2: Method
Method
Development and Improvement
Development
and
85
Improvement
1.1. Introduction When
performing
microcalorimetric
usually relatively high protein low
affinity
In
provide
contaminating On the to
material
hand, this
one
concentrations will be
the macromolecule in
calorimetric
theory,
concentrations
experiments
be
can
required,
it is
exact
dependent
required, particularly in
so
strongly
any
amount
with turbid
concentrations the
on
concentration
will
amount
are to
to additional
recommended to
ligand of
and
is
the
use
of
impurities
or
at
the
are
the
difficult
high protein (proteases,
side reactions with
specific
adequately purified protein.
macromolecule error
on
used. in
the
As
knowledge
AH
is
of
directly
determination
of
its
reliability of this parameter. Moreover, if
be extracted, not
only the protein concentration
but also the amount of active
specific protocols
of
heat effects not due to
strongly dependent
ligand applied,
directly influence
stoichiometric data
exactly known,
of
case
be
samples which
on) may catalyze unwanted
experiments
that
must
trace contamination with other enzymes
even
The outcome of calorimetric the
mind
not interfere with the reactions of interest.
particularly useful
macromolecules, leading
binding. Therefore,
in
keep
high yields. tolerate
can
provided they do
nucleases, phosphatases, and or
should
by spectroscopic methods. On the other hand,
measure
ligands
one
Thus, stable and reliable purification protocols
interactions.
established to
experiments,
must be
protein in the sample. Therefore,
for concentration determination must be established.
86
PART 2: Method
1.2.
Development and Improvement
Expression and Purification
Recently,
in
our
laboratory
the
has been established and
kinetic
measurements
expression
optimized
and
and
for
purification of
recombinant HSV1 TK
obtaining highly pure protein
crystallization
(Bohner,
1996;
Fetzer
However, it has been shown that the kinase is very unstable, and it
develop altered and refined protocols
with
respect
to
suitable for
ef
necessary to
was
stability, quality
1994).
ai,
and
high yield
of the macromolecule.
1.2.1.
Expression
Recombinant
protein
expression
thiogalactopyranoside (IPTG) thrombin
(Fetzer was
being transformed
with the
medium
(LB)
was
plasmid
changed
100
to the
a
the
isopropyl
ß-D-
pGEX2T-TK (Pharmacia) protein
earlier
described
as
were
grown
ug/ml ampicillin.
overnight
The cultures
Gene
at 37°C in Luriawere
expression
was
diluted 1:10 induced
final concentration of 100 uM, After 36 hours for KY895
hours (over night) for BL21, the cells
as a
expression strain BL21 later. After
in fresh medium and grown for 3 hours at 25°C.
addition of IPTG to
using
1994). The TK deficient E.coli strain KY895
plasmid, bacteria
containing
vector
S-transferase fusion
& Folkers, 1992; Fetzer et al.,
used as host, which
Bertani
inducible
glutathione
cleavable
achieved
was
were
harvested by
centrifugation
or
at 4°C
by 20
and
frozen at -70°C.
1.2.2. Isolation of HSV1 TK
The et
protein
was
isolated
mainly according
to a
previously reported procedure (Fetzer
al., 1994), with minor changes. Briefly summarized, the frozen pellet
and
suspended
Triton
and min.
in buffer
(50 mM Tris/HCI pH 7.5,
X-100) before adding
lysed
on
1 mM
(Bohner, 1996). The lysate clarified by
centrifugation
purification
or
at
was
was
|_ig/ml lysozyme
further treated
by
thawed
EDTA, 10% glycerol, 1%
PMSF, 10 mM DTT, 10 mM MgCI2,
ice in presence of 150
Afterwards, the mixture
5 mM
was
1 mM
MnCI2,
and 2000 units DNasel for 15 continuous sonication at 4CC
substituted with 10 mM EDTA to inactivate DNasel,
12,000xg
for 20 min, filtered (0.45
uM) and subjected
frozen at -70°C, where it remained stable for months.
to
PART2 Method
Development and Improvement
87
1.2.2. Purification of HSV1 TK Fusion Protein
HSV1 TK
expressed
was
as
glutathione
S-transferase fusion
protein (GST-TK) and
purified by glutathione affinity chromatography (Hengen, 1996;
LaVallie &
McCoy,
1995; Smith & Johnson, 1988). Formerly established protocols (Fetzer et al., 1994) were
modified since it
(see 4.5.)
was
they
suitable for ITC
were not
experiments.
For
stability
reasons
decided to carry out all measurements with the GST-fusion protein
of HSV1 TK since the kinetic properties
identical to
are
thymidine kinase (Fetzer
et
al., 1994). The
major goal
was
the establishment of
a
strategy that yields pure
protein, free of substrate and cofactor. Therefore, developed. Generally, the
has been
applied
protein by
was
eluted
SDS-PAGE
(PhastGel KY895 fusion
of 67
by addition of
using
Gradient
was
the
5 mM
single-step purification procedure containing
column. After
the fusion protein
thoroughly washing,
glutathione (GSH). Purification
PhastSystem
from
Pharmacia
with
was
was
the fusion
monitored
precasted
gels
10-15) and PhastGel SDS buffer strips. Expression in strain
found to be ineffective, always leading to low expression levels of the
protein,
in the crude extracts, and minor
as seen
kDa) contaminated
as seen
crude extract
glutathione sepharose
to the
a
and active fusion
after
with unknown
purification. Figure
1
(A
yields of fusion protein (MW
impurities (46 kDa and 30 kDa, respectively), and
B) represents typical results of different
batches.
X
Fig.T SDS-PAGE analysis molecular
weights
of
of the marker
the
purification procedure
(lane M) horizontal
glutathione S-transferase (26 kDa). TK 2
(46 kDa)
=
purification protocol yielded
number
HSV1 TK (42
Lane 1 shows crude extract, lane 2
Vertically arranged
kDa),
flow-through
depict lanes, F1
=
from
FP
fragment
1
=
numbers
indicate
fusion protein; GST
(30 kDa)
F2
=
=
fragment
affinity purification (A), (B) Standard
low expression and truncated fusion protein
(A3, B3-5)
88
PART 2 Method
Development
and
Improvement
Fig
2
SDS-PAGE and Western blot
weights
Thrombin
kDa)
TK
analysis
of crude extract
was
(lane
analyzed by
(lane
1 and
M on the left and lanes
1-3)
combination with both antibodies HSV1 TK,
Judged
the molecular
on
in
Lane 1
fusion
antibody
with rabbit
or
fragments
the host
or
F1 and F2
glutathione 2
(46 kDa)
before incubation, lane 2
1
(B) Western
protein from BL21 (lane 2 3 6 and 7) The
against GST only (lanes 5-7)
raised
were
In
originate from GST and
determined to
gels
in
the
and Western blot
second
Thrombin
fragment
This would result
with HSV1 TK
further investigated by
a
origin
was
further
analyzed for
thrombin
and CaCI2 to the protein fraction containing F1
became the
main
fusion protein
cleavage
was
by SDS-PAGE.
site
m
F1
the
achieved
From
Cleavage
product after
including
5 hours
engineered
figure
by adding
2A it is
results first Most
to
fragment
The origin of the
assay
a
thrombin
cleavage
small
a
amount
(Fig 1A, F1), seen
fragment is
thrombin
and
that there
of
was is a
of 26 kDa that
the GST part of the
site and
recognized
parts of the N-
to be
exceptionally
protease cleavage just recently (Pilger, 1999)
findings
fusion protein
in
a
clearly
probably, F1
terminal sequence of HSV1 TK This region has been
susceptible
one
the presence of
site
cleavage
in
thrombin-cleavage
cleavage
monitored for 5 hours
it was
analysis (Fig 2B)
fragment (F1)
The 30 kDa
was
yield,
the fusion protein could be due to protease
due to extract preparation
additional bands
These
fragment
=
of the impurities and the overall low
fragmentation of
a
thrombin
F2
=
5 hours of incubation with thrombin
5) and purified
weight
consisting of GST and
(Fig 2A)
lane 5
protein GST
fusion
(30 kDa)
1
fragment
the left indicate molecular
respectively
concluded that the
activity
=
on
polyclonal rabbit antibody raised against the GST-TK fusion protein
of
means
F1
=
fragment (F1 from Fig 1A)
kDa
3 hours
lane 4
Numbers
purification
depict lanes FP
(42 kDa)
HSV1 TK
=
of the
analysis
vertical number
cleavage of the 30
lane 3, 2 hours
hour
blot
(lane M)
of the marker
S-transferase (26
(A)
^
N
\«^\
are
(FP)
further corroborated and
by Western blot analysis They showed that
fragments descending from
the GST part
(F1) and HSV1 TK
PART2: Method
89
Development and Improvement
Sil?
>i$f0!igäßi':'i
Fig.3:
SDS-PAGE
indicate molecular
~^x
of the
analysis
weights
^iMSSi^XSM"'--^-'
fragment
(30 kDa);
1
The fusion protein is of 66 kDa
F2
of the marker
part (F2) of
new
the fusion
protein
during expression strain
Expression
several unknown
to
affinity purification.
after
Results of
FP
=
protein;
fusion
typical expression using strain
predominant band
on
the
gel
at a molecular
F1
BL21.
weight
present in the crude
are
is not
mainly due
extract and the
purified protein
purification protocol, but
to the
in the host.
KY895
proteases. This let
lanes. Lane 1 shows crude
is shown in lane 3 and 4.
(Fig.2B). Obviously, proteolysis occurs
a
depict
numbers
protein of -70% purity (lane 3). (B) The quality of fusion protein
the
procedure
vertical number
flow-through
highly expressed, forming
(lane 1-2). Elution yields
achievable with the
(lane M),
(46 kDa). (A)
2
fragment
=
^^;:i:V:i^V:50iï^/:ï:ËlfÏ!ï;o'•';;'i:ï
:
optimized purification procedures. Horizontally arranged
extract after the isolation process, lane 2 =
^'-'^Hï.::.:''.
:-:vs-:
changed
was
high yields
BL21
to
of fusion
proteins (Fig.3A), amongst
protein
which that
deficient
is
in
several
still contaminated
was
by
which the GST part and the HSV1 TK
part of the fusion protein could be detected by Western blot analysis (Fig.2B).
Subsequent contamination
the
purification
let to about 80% pure fusion
quantified by gel densitometry (Fig.3B)
following:
the crude extract is
washed with buffer A
glycerol, finally,
1% Triton
the
protein
mM Tris/HCI
(50
X-100), was
applied twice
protein
as
The final to the
pH 7.5,
to
respect
protein
detected
by SDS-PAGE
purification procedure
was
glutathione sepharose column, NaCI,
4 mM
containing
10 mM
150mM
then washed with buffer A
EDTA, 10%
MgATP,
and
directly (on-column) exchanged into the experimental buffer
by thoroughly rinsing the column addition of 5 mM GSH and
yielded about
with
protocol
through unspecific column interactions, proteolytic cleavage and DnaK
co-purification, finally and
of
optimization
10 to 20 mg
was
with
excess
directly
of buffer. The
used for titration
protein per liter of culture.
protein
was
eluted
by
experiments. This protocol
90
PART2: Method
1.2.3.
Expression
was
available in
Briefly,
strain
binding
protocol had
no
high
studies in respect of
(ODeoo)
Improvement
periplasmic
domain of the histidine
to be established since pure and stable
BL21, harboring expression plasmid pET-CitAP, served
The cultures
reached
grown in
were
LB
medium
incubated at 37°C until the
were
value between
a
protein
amounts.
overproduction of CitAPHiS and
kanamycin.
and
and Purification of CitAPms
For the calorimetric
autokinase CitAP,
Development
0.6 and 0.8.
as
containing
optical density
Expression
host for
at 600 nm
induced
was
ug/ml
50
by
the
addition 1 mM IPTG and the cultures were incubated for another three hours at 30°C.
Subsequently, cells mM Tris/HCI For
were
pH 7.9,
harvested
NaCI) containing
500 mM
disruption, the
cells
were
supplemented with
0.25
mg/ml
EDTA-free)
at
the
by centrifugation, washed
in the
resuspended DNasel and
concentration
5 mM
a
Aminco)
108
at
centrifugation (30
min
ultracentrifugation (1 supernatant desired
CitAPHis
buffer
same
27000xg, 4°C).
at
h
150000xg,
at
passed through
was
purified by Ni2+
for CitBHiS
(wet weight)
containing
chelate
(Meyer
was
ef
by
the
supplier
French pressure cell
a
debris
cell
were
The cell-free extract
4°C) urn
to
the
sediment
(Boehringer
was
removed
by
subjected
to
membranes.
The
filter and used for the isolation of the
5 mM imidazole.
five bed volumes of buffer A
was
a
column with 2 ml (bed
with buffer A
Weakly
(20
bound
containing
400 mM imidazole in buffer A.
filtration with
affinity chromatography essentially
mM Tris/HCI
proteins
were
volume)
pH 7.9,
removed
30 mM imidazole. Elution
Subsequent
buffer
exchanges
Sephadex G-25 (PD-10 columns, Pharmacia). monitored
as
described
ai, 1997). The soluble fraction obtained from 1-2 g
loaded onto
(Novagen) pre-equilibrated
proteins
0.2
a
and
cells
weight)
wet
protease inhibitor cocktail (Complete,
recommended
intact
(4 ml/g
proteins.
previously cells
was
MPa,
(20
imidazole, and stored at -20°C.
Mannheim). After passing the cell suspension twice through
(SLM
in buffer A
once
by SDS-PAGE (Laemmli, 1970)
and
500 mM
The
NaCI)
by washing
with
performed
with
was
were
His~bind resin
performed by gel
purification
staining
of all
with Coomassie
brilliant blue. This
protocol yielded about
(>98%).
10 to 20 mg
protein per g
cell
weight of high purity
PART2: Method
91
Development and Improvement
1.3. Concentration Determination Procedures 1.3.1. Concentration of HSV1 TK
Since the acid
protein
analysis (Gill
standard
&
curve on
von
Hippel, 1989),
and it
the basis of the fusion
Therefore,
concentrations
protein
(Bradford, 1976) impurities
with bovine
serum
possible
to establish a
precise
protein. Under these circumstances, it is
protein
estimated
were
albumin
by SDS-PAGE
detected
amino
accomplished by
not be
was not
difficult to estimate the amount of active
even more
for
impure, concentration could
was
as
in the
using
sample.
dye-binding
a
standard. The content
was
assay
corrected
quantified by gel densitometry using
and
a
CAMAG Electrophoreses Scanner II with CATS software.
1.3.2. Concentration Determination of CitAPHiS The
protein
amino
was
acid
determined
essentially pure
analysis
(Gill
&
and concentrations could
Hippel,
von
spectrophotometrically
CitAPHis
1989).
using
at 276 nm
e
easily
=
be calculated
concentrations
by
were
7,25 mM"1cm"1.
1.3.3. Concentration of dT and ATP
Determination of dT and ATP
spectroscopic final
methods. For
ligand solutions,
straightforward
was
experiments
concentrations
where both dT and ATP
were
determined
1.3.4. Concentration of Citrate and Citrate
Citrate
was
calculated
directly
on
were
be done
present in the
HPLC assay (see
an
by
4.6.)
Analogs
further
purification,
and
concentration
were
the
Measurements
Activity
purification protocol
which
without
by
easily
by weight.
1.4. Kinetic and The
used
and could
led to
dT/enzyme
interaction
Furthermore, the experiments
activity of HSV1
samples containing
TK. To date,
were
no
data
was
tested based
planned were
5 mM
to take
glutathione, on
place
the influence of
kinetic measurements. at the
available with respect to
pH of
maximum
pH dependence
92
PART2: Method
of the fusion
protein. Consequently, these parameters
radioactive enzyme assay This assay is based a
on
previously
3H-dTMP
digestion
of the
on
the paper
DEAE
paper
are
with
3H-dT
& Folkers,
(Furlong, 1963)
a
1996). which represents
anion-exchange
paper retains
to be washed away, The reaction
counted in
cellulase
investigated using
were
(Gerber
nucleoside kinases. The
but allows the unreacted
adsorbed
products
established
the DEAE-cellulose method
rapid assay technique for
labeled
Development and Improvement
scintillation counter after
liquid
a
prior
scintillation
to
counting.
This
procedure is especially useful if solution conditions like pH, salt, buffer substances have to be
changed.
1.4.1. Effect of Glutathione
The effect of GSH
on
reciprocal analysis of
the kinetics of HSV1 TK at 37°C
initial
velocity data obtained
5 mM
determined, corresponding very well
to
Vnax,
(Vmax
=
1.4.2.
in literature is 0.2 uM no
493
pH 7.2, 0.2%
ATP, 5 mM, 5 mM MgCI2 and 10 ng/ml purified cleaved HSV1 TK.
For measurements in presence of 5 mM GSH
reported
pmolmg"1min"1)
Km value of 0.18 u.M has been
a
to the value of 0.14 uM without GSH
(Chen
significant difference
was
ef
ai, 1979; Michael
et
(Fig.4).
ai, 1994). With respect
found for reactions in presence of 5 mM GSH
and without GSH
(Vmax
=
443
pmolmg"1min"1).
pH
on
thymidine kinase activity
was
studied
by varying
the
reaction buffer with 100 mM Tris/HCI in the range of 6.5 to 9.5
(Fig.5).
mixtures contained 0.175% BSA, 5 mM ATP, 5 mM
18
pH
protein. Since
the
buffering capacity
of the actual solutions
The
was
is low
MgCI2
beyond pK
+
and 1
reaches
a
maximum between
sharply. Suitable
pH of
the
Reaction
ng/ml purified
(pKa Tris/HCI
=
8.0),
controlled at 37°C.
catalytic activity of HSV1 TK of fusion protein increases from pH
Thus,
Km
pH-Dependent Activity
The effect of
fusion
double
with variable concentrations of dT
from 20 uM to 800 uM. The reaction mixture contained 50 mM Tris/HCI
BSA,
by
evaluated
was
pH
conditions for
7 to 8. At
calorimetry
measurements were carried out at
pH
higher pH values, would 7.5.
be at
the
pH of
6.5 to 7.5 and
activity decreases maximum
activity.
PART2- Method
-20
-10
10
Development and Improvement
20
30
40
50
93
60
1/[dT] Fig.4: Kinetics GSH
given
of substrate
(filled squares)
phosphorylation
of recombinant HSV1 TK fusion
and in presence of 5 mM GSH
in uM. The solid lines
(open circles),
represent linear regression fits
and 0.18 ijM without and in presence of
to the
v
is
given
as
protein without added
1/prnol,ug 1min \
dT is
date, yielding Kt11 for dT of 0.14 ,uM
GSH, respectively.
120 100
80 o
60
.5:
40
Œ
20
8
10
pH Fig.5:
Effect of
pH
on
thymidine
kinase
activity of HSV1 TK fusion protein measured
Fusion protein exhibits maximal activity in the pH range of 7 5 to 8.
at 37°C.
The
94
1.5.
PART2: Method
Stability
1.5.1. Time
Measurements
Dependent Activity
For rapid and easy
to
of
oxidation
phosphorylation
screenings
a
enzyme
(Gerber, 1997)
been established due
Development and Improvement
NADH
coupled UV-spectrophotometric assay
which monitors the time
and
corresponds
the
to
dependent change
ADP
formation
Activity
the
reaction. The method has limitations since it is associated with
measurements were carried out at 37°C
containing
in A340
during
enzyme cascade that prevents modifications of the reaction conditions such
50 mM Tris/HCI
phosphoenol pyruvate,
pH 7.2,
5 mM
MgCI2,
with 75 ul
1 mM
of
a
has
an
pH.
as
reaction mixture
DTT, 0.3 mM NADH, 0.4 mM
0.45 U pyruvate kinase, 0.5 U lactate
dehydrogenase,
1 mM
dT, 160 uM ATP, and 1.7 ug fusion protein. No loss of activity could be detected for
Afterwards, the catalytic activity decreased sharply
at least 20 hours.
value of 20% of the
starting
to a residual
conditions.
120
g
100
.e >
80
B
40
CC
20 0 100
50
150
Time(h) Fig.7: Decrease
in
50 mM Tris/HCI
pH
could
activity 7
of HSV1 TK fusion
at 25°C as a function of time. Conditions
5, 4 mM EDTA, 5 mM GSH, 1 mM DTT. For at least 24 hours,
be detected, thus the
sharply
protein
after 30 hours to
a
enzyme remained stable under these conditions.
residual
activity of 20% of the starting value.
no
were:
loss in activity
Activity decreased
PART2. Method
Modern calorimeters allow to
70° C
which
dependence of fundamental
95
Stability
1.5.2. Thermal
5°C
Development and Improvement
precise be
can
AH
or
the
assumption
measurements in the
exploited
are
form of the
biological
of
the
method
is
that
the
experiments
from
are
the
measured
thermodynamic
of the
assumption
conducted since
temperature
temperature. The
with
ligand binding equilibrium
macromolecules. However, this
whenever calorimetric
ACp
calculate
thermodynamic linkage of KB
the resultant of the
parameters
to
temperature range of about
proteins
natively folded
should be tested
unfold at
higher
temperatures. Therefore the thermal nm
stability of HSV1 TK
by circular dichroism
unfolding
occurs
with
a
as
a
was
studied
by monitoring ellipticity
function of temperature.
Figure
Tm of 42°C with transition starting
near
8 shows that
at 217
global
35°C. The maximum
temperature useful for the present ITC binding study would be about 30°C.
i
5.0E+05
0.0E+00
è
-5.0E+05
-
--
-
o
S Q.
-1.0E+06 -1.5E+06
-
-
UJ
-2 0E+06
-
2
-2.5E+06
-
-3 0E+06 15
25
35
45
55
65
75
Temperature [°C]
Fig.8:
Thermal
scan rate
of 0.4
of 20°C/h. Conditions were 10 mM Tris/HCI
mg/ml.
the maximum
Wurth).
unfolding of HSV1 TK monitored by circular dichroism. Measurements
The
signal
pH
is normalized to the monomer
useful temperature for titration
7 5, 1 mM EDTA and
Unfolding begins
experiments
as
30°C
a
were
done at
a
protein concentration
to occur near
35°C, giving
(figure kindly provided by Ch.
96
PART2 Method
1.6.
Development
High Performance Liquid Chromatography
High performance liquid chromatography of
Improvement
and
thymidine and
ATP
final
the
in
was
applied for concentration determination
ligand solutions,
and
phosphorylation products during calorimetric experiments based
on
published
chromatography
ion-pair
(Masson
method
dTDP and dTTP in
a
single
et
using
al., 1993). It
run
is
a
modified
monitor
to
HPLC
The
protocol of
a
potential system
is
previously
able to separate ADP, ATP, dT, dTMP,
(Fig 5)
öS „£ r
ii
i
i
f
f .„^l
Ü
U
J
u
l
M
I
I
I
!
li
!
I
I
I
I
I
)
'
I
l>
M I
I
Fig
8
HPLC system
formation
during
for monitoring
substrate catalysis
ADP
and
nucleoside
I
!
M
i
i
mono-
I
I '
1» '<
I
I
!
t
I
I
I
!
'
M>
>,(>
'I
It
diphosphate
i
and
triphosphate
PART2: Method
Nucleotides
C18 column
100 RP-18, 5 urn, 250x4 mm,
M NaH2P04, 25 mM
phase (0.2 a
(LiChrospher
tetrabutylammonium,
rate of 1.0 ml/min and detection at 254
concentrations
solutions
were
showing linearity
calculated
Ligand
(v/v) methanol)
delivered at
by
means
of calibration
curves
from standard
in the range of 99% of a
1:1 mixture of 1.6
PART 3:
1
OH
Order of Substrate
Compulsory
•
1
F"
'
1
/
i
| If
-2
-4 -6
'
I
t
u
V
y
'
j
'
I
to HSV1 TK...
Binding
v
I
»
»
'
'
1
I
-
-
*
111
A •—-
B 0.0 0) CO
0) a a.
3.
0.5
i
0-1
v*""y%V"",V""
1.0 '-
1
i
i/ T'T
if
1.5 '
i
•
i
2.0
i
'
i
'yuyy'\r,,Y^r'\ry
r
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-
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i
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;
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159
m
m
Domain of the Histidine
Autokinase CitA Functions
as a
Highly Specific
Citrate Receptor
Sibylle
Kaspar1,3,
Leonardo
Remo
Scapozza2,
'Mikrobiologisches
Perozzo2,
and Michael
Institut,
Reinelt2, Margareta Meyer1,
Stefan
Karin
Pfister1,
Bott1,3
Eidgenössische
Technische
Hochschule,
Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland; Departement Pharmazie, Eidgenössische Switzerland;
Technische
3Present
Hochschule,
address:
Institut
Winterthurerstr.
für
190,
CH-8057
Biotechnologie I, Forschungszentrum
Jülich, D-52425 Jülich, Germany
Dedicated to Professor Rudolf K. Thauer
Molecular
Microbiology 33(4),
858-872
on
the occasion of his 60th
(1999)
Zürich,
birthday.
160
3.1.
PART 3: The
Periplasmic
Domain of the Histidine Autokinase CitA...
Summary
The two-component
regulatory system CitA/CitB
is essential for induction
citrate fermentation genes in Klebsiella pneumoniae. CitA represents bound
kinase
sensor
consisting
transmembrane helices, domain. A fusion the
regulator CitB. and
domain, and the conserved kinase
y-phosphoryl group
The autokinase
phosphorylation
of CitB
and D56 represent the
activity of CitA
was
inhibited
phosphorylation
presence of ATP, CitB-D56N formed the sensory
properties of CitA,
overproduced tag
as a
(CitAPHis)-
Purified
by isothermal
with
T»S
The
dianionic
presence of that
the
the
pH-dependency is
the
citrate
domain of CitA as
not
at
of
none
pH 7)
in
45 to
a
the
binding
analyze
176)
was
histidine
other
tri-
was
change
driven
was
and
by
As the
opposed (-
reaction indicated that the
species recognized by CitAPHiS-
bound
In the
stoichiometry.
1:1
reaction
the entropy
of the
In
the
significantly increased, suggesting
by CitAPHiS-
highly specific
a
with MalE-CitAC. To
C-terminally attached
binding
ions the dissociation constant is
a
but
\iM
5
exchange,
respectively.
periplasmic domain (amino acids
~
response
exchange, indicating that H350
complex
kJ/mol), whereas
-76.3
H-citrate2"
Mg2+
D56N
a
stable
calorimetry,
Mg-citrate complex
periplasmic
binding
kJ/mol).
form
two
transmitter
or
H350L
an
sites of CitA and CitB,
high affinity (KD
titration =
+46.3
the
a
by
by
abolished
was
CitAPHls bound citrate,
enthalpy change («H =
by
of ATP to its cognate
soluble, cytoplasmic protein with
dicarboxylates tested, shown
flanked
domain
(amino acids 327-547) showed constitutive autokinase
transferred the
and
periplasmic
a
membrane-
a
protein (MalE-CitAC) composed of the maltose binding protein and
CitA kinase domain
activity
linker
a
of
of the
This
work
defines
the
citrate receptor and elucidates the
characteristics of CitAPHiS.
3.2. Introduction
Regulation
of bacterial gene
often controlled
expression in response
to environmental
by two-component regulatory systems, consisting of
and a response
regulator. The
autophosphorylation
of
a
subsequently transferred which then mediates
sensor
kinase
responds
to
a
conserved histidine residue, and the to
a
changes
a sensor
in gene
expression
or
kinase
certain stimulus by
phosphoryl group
conserved aspartate residue of the response
changes
is
cell behavior
(for
is
regulator,
reviews
see
PART 3: The
Periplasmic Domain
Bourret et ai, 1991; Parkinson & In
the
of
majority
extracellular N-terminal
Kofoid, 1992; Stock kinases
sensor
cases,
sensor
of the Histidine Autokinase CitA...
are
(typically
domain
flanked by two transmembrane helices and domain a
(-220
amino
of variable
region
linker
acids) that is connected
length.
an
et
161
ai, 1989; Stock et ai, 1995).
transmembrane
with
proteins
an
length)
100 to 200 amino acids in
intracellular C-terminal autokinase
to the second transmembrane helix via
This architecture enables the
proteins
to sense
external stimuli and transduce information to the cytoplasm. Results obtained with several the
kinases have shown that these
sensor
autophosphorylation
occurs
(for
in trans
many two-component systems have been cases
the
primary signals recognized by
membrane-bound
its
Mg27Ca2+-responsive resembles that of the
CitA from response
genes,
as
we
sensor
have
Klebsiella
which is
in the past years,
in
a
few
by the difficulties in purifying
overcome
this problem is to express to
examples of this approach represent the (Vescovi
Koshland,
the
ai, 1997),
et
1993),
whose
or
the aspartate
modular
structure
and the kinase domain of the
periplasmic
is essential for the
expression
that citA and citB mutants
sensor
two
divergently
of citrate
of the citrate fermentation
were no
sole carbon and energy
as
a
cluster
longer able source
on
to grow
(Bott, 1997;
the chromosome,
transcribed units, the citC operon and the citS
operon. The citC operon encodes citrate
(citF)
only
kinase have been identified
sensor
1995). The citrate fermentation genes form
and a-subunit
Although
pneumoniae. This protein, in cooperation with its cognate
by the fact
composed of
Stock et ai, 1995).
kinases described above.
analyzed
regulator CitB, shown
D.E.
&
dimers and that
as
separate, soluble protein, which then allows
a
under anoxic conditions with citrate Bott et ai,
given
a
kinase PhoQ
sensor
(Milligan
chemoreceptor
as
Successful
in vitro.
properties
In this work,
see
analyzed
kinases. A strategy to
sensor
extracytoplasmic domain
study
review
In part, this lack of information is caused
biochemically.
the
proteins function
lyase, and
lyase ligase (citC), the a
y-
protein (citG) presumably
(citD), ß- (citE), involved in the
biosynthesis of the 2'-(5"-phosphoribosyl)-3/-dephospho-CoA prosthetic group of citrate
lyase (Bott & Dimroth, 1994).
citrate carrier CitS
Na+-pump
(citS),
oxaloacetate
and the y-
(oadG),
decarboxylase.
genes of the citS operon and sensor
The citS operon encodes the
are
The
a-
(oadA),
and
citAB genes
Naf-dependent
ß-subunit (oadB) are
the
positively autoregulated (Bott
et
of the
promoter-distal ai, 1995). The
kinase CitA shows the typical modular composition described above, i.e. it
162
PART 3: The
Periplasmic Domain
of the Histidine Autokinase CitA.
.
kinase domain
..-A^, 1
periplasmic
1
domain
44
24
linker
TMH2
CitAPHIS (139 amino acids,
15.3
Fig. 1. Domain organization of the
(TMH1 and TMH2),
the five
316
Q1
signature
two
amino acid
kDa)
sensor
segments
kinase CitA from K.
the different features within the
primary
(Fig. 1).
boxes
sequence
The
analysis
composed of
ATP-binding
confirmed
of the »-subunit of oxaloacetate with
the
plasmid
by
shown. The
shown
are
phosphorylation
in the
course
plasmid.
sequencing
Analysis
of transposon insertions
In
one
and found
to
case,
be
the
exact
of
In
subdomain with the
a
predicted from
topological analysis ai, unpublished)
ef
this
Purified,
a
resulting
were
site
located
transcriptional
conserved N-terminal receiver domain and
motif.
fusion
after Thr-47,
transmembrane helix of CitA. The
helix
linker
N-, G1-, F-, and G2-
decarboxylase (Di Berardino
phosphatase activity revealed that the majority of them the
a
study,
a
target
used that contained besides the oadB gene also the 5'-terminal part of
citA up to codon 121.
of
helices,
the N-terminal domain
accidentally
in the lower
schematically.
two transmembrane
the
are
by the numbers,
TnphoA transposon (Manoil & Beckwith, 1985). was
The two transmembrane
Kofoid, 1992)
subdomain with the
periplasmic location of
was
acids, 66.8 kDa)
and G2-box
structure is indicated
domain flanked
and the kinase domain
conserved H-box and the
amino
H545H546G54
(named H-, N-, G1-, F-,
Parkinson and
part, the proteins CitAPHiS and MalE-CitAC used in this study
region,
pneumoniae.
of the kinase domain
characteristic amino acids;
periplasmic
G1 F G2
N
(604
547
putative Q-linkers (Q1 and Q2; Wootton and Drummond, 1989), and
position of
a
H
MalE-CitAC
to the
consists of
442 480 492 505
MalE(A2-26)-la7Na28Ta2,
according
most
subdomain
331 350
Q2
'
ATP-binding
subdomain
Q,73L174E175-HHHHHH
M-D45I46T48
helices
region
178 199 2 4 229
TMH1
phosphorylation
_
was
in strong alkaline
located in the citA part determined
by
DNA
immediately behind the first
activator CitB is
composed
C-terminal domain with
unphosphorylated CitB binds specifically
to
a
the
of the
helix-turn-
citC-citS
PART 3: The
intergenic region,
Periplasmic Domain
but with low
changes, resulting
in
an
affinity.
of the Histidine Autokinase CitA...
In vitro
increase of its
phosphorylation of CitB
binding affinity by
a
163
elicits structural
factor of -50
(Meyer
et
ai, 1997). the citrate fermentation genes has to be
Expression of
synthesis of both citrate lyase conditions
could
triggering
futile
a
and oxaloacetate
severely affect
the
cytoplasmic membrane.
the
anaerobic conditions as a
periplasmic
of
the
citric
(Bott
ef
as
a
acid or
Previous studies showed that
inappropriate either
by
by deprivation
of
cycle,
we
expression
was
proposal by analyzing the
tested this
were
or
that CitA may
proposed
separate protein. Moreover, the properties of
autokinase domain and its interaction with CitB
of the
citrate, Na+ ions, and micro-
on
ai, 1995). Therefore, it
citrate sensor. In this work,
domain
under
since the
decarboxylase could perturb the Na+ balance
citC operon and the citS operon is dependent
function
decarboxylase
cycle of citrate synthesis and cleavage
oxaloacetate. In addition, oxaloacetate across
function
carefully regulated,
the
CitA
analyzed.
3.3. Results 3.3.1. Isolation and Characterization of the CitA Kinase Domain As
part of
our
efforts to understand the mode of action of the two-component
protein CitA from Klebsiella pneumoniae, the CitA kinase
547)
was
peptide.
fused to the C-terminus of the maltose
The
resulting
Escherichia coll and
fusion
domain
acids 327
binding protein lacking
protein MalE-CitAC (66.8 kDa)
was
Fig. 2,
contained
predominantly
the fraction obtained after elution with a
-
signal
resin.
maltose-containing
in
As
buffer
protein of the expected size of 66 kDa, but also several
in the range between 65 and 40 kDa and another
27 kDa. Whereas the latter
its
overproduced
purified by affinity chromatography using amylose
shown in
degradation products
(amino
sensor
corresponds
to the CitA
one
of about
part of MalE-CitAC, the former
presumably consist of the entire MalE (-40 kDa) plus different proportions of the CitA kinase domain. Further
column in
a
with
purification
of MalE-CitAC
by gel
on a
Superdex-200
(Pharmacia) revealed that generally about 80-90% of the protein
multimeric state of >600 kDa, whereas 10-20% a
filtration
size of 130 kDa
(data
not
larger aggregates formed again
was
shown). Upon storage
as
shown
present in
a
was
present
dimeric state
of the dimeric form at 4°C,
by native PAGE (Fig. 2,
lane
7).
164
PART 3: The
kDa
Periplasmic Domain
2
1
3
of the Histidine Autokinase CitA...
4
5
6
7
97.2 66.4 55.6 42.7
36.5
26.6
20.1
Fig.
2.
Overproduction, purification and functional analysis
standards. Lane 2 and 3, whole cell lysates of E. coli
of MalE-CitAC. Lanes 1
DH5*/pMalE-CitAC
and 4,
protein
before and 3 h after IPTG
induction, respectively. Lane 5, MalE-CitAC (15 ug) obtained after affinity chromatography of cell
amylose resin. Lane
extract on
1
to 5 show Coomassie-stained
autophosphorylation of MalE-CitAC (0 5 ug) after [" P]-ATP (30000 dpm/pmol) Lane 7, native PAGE
(~
5
ug)
obtained after
as
revealed
(Laemmli system) showing Coomassie-stained storage of the dimeric form at 4°C for
autophosphorylation
analysis (Fig. 2,
lane
as
shown
phosphorylated. Analysis
filtration
revealed that the
According proposed directed
to sequence
to be the
alignments
the
amylose chromatography
of the
y-[32P]-ATP
with
of the MalE-CitAC multimer
to the dimeric form
with other histidine
showed the
protein
formed
a
same
result
(data
not
had
was
significantly
shown).
et
was
was
ai, 1995). By sitewas
replaced by
a
overproduced and
of the protein obtained after described for
multimer of >600 kDa and
present in the dimeric form. MalE-CitAC-H350L
a
protein kinases, His-350
in MalE-CitAC
as
27 kDa also
complexes obtained after gel
(>600 kDa) usually
corresponding residue
led
subsequent Phospholmager
and
described for MalE-CitAC. Gel filtration
major part
week.
resulting protein MalE-CitAC-H350L
amylose chromatography the
multimeric forms of MaiE-CitAC
autophosphorylated residue of CitA (Bott
mutagenesis,
as
one
by SDS-PAGE
activity compared
leucine residue. The
purified
large
6,
temperature with 20 uM y-
6). Several degradation products including that of
became
weaker autokinase
10 min incubation at room
Lane
by SDS-PAGE and subsequent Phospholmager analysis.
Incubation of the protein obtained after to
SDS-polyacrylamide gels.
not
a
MalE-CitAC, i.e. minor part
autophosphorylated
was
in the
PART 3: The
presence of
Periplasmic
Domain of the Histidine Autokinase CitA...
y-[ P]-ATP, providing strong support
for the
assumption
165
that His-350
represents the phosphorylation site of CitA.
3.3.2. CitB The
Phosphorylation by MalE-CitAC
ability of the CitA kinase domain
MalE-CitAC and CitBHiS, its C-terminus
including
CitAC transferred the of MalE-CitAC,
velocity
of
no
CitB derivative
tested in vitro with
ai, 1997). As shown in Fig. 3, MalE-
ef
efficiently
ATP
of CitBHiS with
phosphorylation
was
additional amino acids at
containing eight
(His)6 tag (Meyer
a
phosphorylation
(data
phosphorylate CitB
y-phosphate group of
CitBHiS
concentrations terminal
a
to
to
CitBHis. In the absence
y-[32P]-ATP
increased
with
observed. The
was
MalE-CitAC
increasing
shown). Besides CitBHjS also CitBNHis which lacks the C-
not
DNA-binding part of
CitB
(amino acids 139-242)
phosphorylated by
was
MalE-CitAC. This result shows that the kinase domain of CitA (amino acids 327-547) and
the
determinants transfer.
domain
receiver
Exchange
of the no
that
only
be
Asp-56 represents
(about 3-fold; data
could
be
by
not
an
(Fig. 4,
Therefore, citrate more
this
was a
specifically, by
hypothesis,
we
on
the
phosphoryl
led to
alignments (Bott
sequence
(1.5-
interaction of the two was
able to form
(Fig. 4,
CitBHiS-
et
ai,
5-fold; Fig. 3) and the
to
proteins. Analysis by native a
stable
lanes 5 and
to a minor extent and
complex with MalE-
6). CitBHis also formed
apparently
a
not influenced
lanes 3 and 4).
and Purification of the
specifically required
and
structural
shown) of MalE-CitAC autophosphorylation. This effect
The CitA/CitB two-component genes
interaction
against asparagine
stimulated the extent
MalE-CitAC, but only
Overproduction
the
all
phosphorylation site of CitB. Interestingly, the
the
in the presence of ATP
the presence of ATP
3.3.3.
1-138) carry
phosphorylated in vitro by MalE-CitAC (Fig. 3). This
usually
explained by
CitAC, especially with
acids
residue in CitBHiS
PAGE revealed that CitBHls-D56N
complex
(amino
the prediction based
presence of CitBHiS-D56N rate
Asp-56
longer
strongly supports
1995)
CitB
required for the specific protein-protein
D56N, which could result
of
Periplasmic
regulatory system
Domain of CitA
is essential for the induction
of
for the anaerobic catabolism of citrate in K. pneumoniae.
likely target
to be
recognized by
periplasmic part of
this
tested whether this domain is
the
protein (Bott
sensor
et
kinase CitA,
or
ai, 1995). To verify
capable of binding
citrate. For this
166
PART 3
The
Periplasmic Domain of the Histidine Autokinase CitA
123456789
3.
Fig.
MalE-CitAC
MalE-CitAC
CitBHls (lanes 4-6)
CitBHls-D56N (lanes 7-9)
Phosphorylation
of MalE-CitAC
The three assay mixtures
(-1000 dpm/pmol)
ATP
uM
(total
in
the absence and
volume 50
and either
no
ul)
8),
and
SDS
temperature 15 ul aliquots
other protein
and 15
mm
(lanes 3,
loading buffer
PAGE and the dried
6 and
were
9)
and stored on ice
gels
were
12
reaction
4
by
immediately
Subsequently
analyzed
3
(lanes 1-3)
with
5
a
or
9 4 uM
CitBHs
(dimer)
or
CitBHs-D56N
0 5 mM
CitBH
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