from one domain to the other within the molecule, the role of substrates in Keywords domain closure; phosphoglycerate kinase; molecular graphics; substrate effect; thermal unfolding Corr
Trang 1ternary enzyme–substrate complex and domain closure
of 3-phosphoglycerate kinase
Andrea Varga1, Bea´ta Flachner1, E´va Gra´czer1, Szabolcs Osva´th2, Andrea N Szila´gyi1
and Ma´ria Vas1
1 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary
2 Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
The experimental and theoretical studies that led to
our present understanding of protein structural
chan-ges and their role in enzyme function were mostly
carried out on small single-domain proteins [1] Most
enzymes, however, are built of several domains The mechanism of transmission of molecular interactions over large distances (e.g from one domain to the other) within the molecule, the role of substrates in
Keywords
domain closure; phosphoglycerate kinase;
molecular graphics; substrate effect;
thermal unfolding
Correspondence
M Vas, Institute of Enzymology, BRC,
Hungarian Academy of Sciences, H-1518
Budapest, PO Box 7, Hungary
Fax: +36 1466 5465
Tel: +36 1279 3152
E-mail: vas@enzim.hu
(Received 18 December 2004, revised 15
February 2005, accepted 17 February 2005)
doi:10.1111/j.1742-4658.2005.04618.x
3-Phosphoglycerate kinase (PGK) is a typical two-domain hinge-bending enzyme with a well-structured interdomain region The mechanism of domain–domain interaction and its regulation by substrate binding is not yet fully understood Here the existence of strong cooperativity between the two domains was demonstrated by following heat transitions of pig muscle and yeast PGKs using differential scanning microcalorimetry and fluorimetry Two mutants of yeast PGK containing a single tryptophan fluorophore either in the N- or in the C-terminal domain were also studied The coincidence of the calorimetric and fluorimetric heat transitions in all cases indicated simultaneous, highly cooperative unfolding of the two domains This cooperativity is preserved in the presence of substrates: 3-phosphoglycerate bound to the N domain or the nucleotide (MgADP, MgATP) bound to the C domain increased the structural stability of the whole molecule A structural explanation of domain–domain interaction is suggested by analysis of the atomic contacts in 12 different PGK crystal structures Well-defined backbone and side-chain H bonds, and hydropho-bic and electrostatic interactions between side chains of conserved residues are proposed to be responsible for domain–domain communication Upon binding of each substrate newly formed molecular contacts are identified that firstly explain the order of the increased heat stability in the various binary complexes, and secondly describe the possible route of transmission
of the substrate-induced conformational effects from one domain to the other The largest stability is characteristic of the native ternary complex and is abolished in the case of a chemically modified inactive form of PGK, the domain closure of which was previously shown to be prevented [Sinev MA, Razgulyaev OI, Vas M, Timchenko AA & Ptitsyn OB (1989) Eur J Biochem 180, 61–66] Thus, conformational stability correlates with domain closure that requires simultaneous binding of both substrates
Abbreviations
1,3-BPG, 1,3-bisphosphoglycerate; 3-PG, 3-phospho- D -glycerate; CM-PGK, carboxamidomethylated PGK;DSC, differential scanning
microcalorimetry; GAPDH, D -glyceraldehyde-3-phosphate dehydrogenase; PGK, 3-phospho- D -glycerate kinase or ATP:3-phospho- D -glycerate 1-phosphotransferase; W122, yeast PGK with mutations of Y122W, W308F and W333F; W333, yeast PGK with mutation of W308F.
Trang 2this process, and the fulfilment of enzyme activity
through these structural changes remain to be
elucidated
3-Phosphoglycerate kinase (PGK; EC 2.7.2.3) is a
typical two-domain hinge-bending enzyme [2–5] with a
conserved primary structure [6] and tertiary fold [2–
5,7–9], including a well-structured interdomain region
PGK is therefore a suitable model with which to study
the mechanism of domain–domain interplay and its
role in both protein stability and function In order to
understand how the two substrates affect domain
interplay and thereby induce the hinge-bending type of
domain movement, the effect of the substrates (both
separately and together) on PGK conformation needs
to be clarified
Substrate-induced stabilization of PGK structure
against chemical modification [10–15], proteolytic
de-gradation [16,17] and unfolding [12,18–20] has been
observed Substrate-induced conformational changes
have been detected by various techniques,
inclu-ding NMR [21–23], fluorescence [15,24–29] and
ESR-spectroscopy [30], analytical ultracentrifugation [31],
small angle X-ray scattering [32–34] and X-ray
crystal-lography [3,4,8] There are, however, conflicting results
concerning the requirement of either only one or both
substrates in causing domain movement and the
ques-tion of whether binding of a single substrate to one of
the two domains can also stabilize the structure of the
other domain [32–34] Often the results of studies with
the solubilized PGK do not support the suggestion
from the X-ray crystallographic work about the
requirement of both substrates (i.e formation of the
ternary enzyme–substrate complex) for domain closure
The contradiction may be attributed to prevention of
occurrence of the large-scale movement of domain
clo-sure by the lattice forces operating in certain PGK
crystals [35]
The best approach by which to obtain direct
infor-mation about the extent of domain–domain
coopera-tivity, is to carry out unfolding–refolding experiments,
as noninteracting structural domains generally
corres-pond to separate folding units Both unfolding [36–50]
and refolding [36–38,46–62] experiments using
denatu-rants have been carried out extensively with PGK, but
mainly in the absence of substrates, with the intact
enzyme [36,37,39,41,43,44,46,48], its engineered mutants
[39–41,46,55,62] and its various molecular fragments
[40,50–55,57–63] These studies show that the two
domains exhibit slightly different stabilities and
unfold-ing⁄ refolding of the two domains probably occurs in a
sequential order within the PGK molecule No
com-prehensive picture, however, has yet emerged about
the relative stability of the two domains and about the
extent of domain–domain interactions, especially not
in the enzyme-substrate complexes
Heat- or cold-induced unfolding of yeast [19,20,64– 73], thermophile [64,65,74,75] and cold-active [76] PGKs in the absence [64–75] or presence [19,20,76] of substrates has been monitored by differential scanning calorimetry (DSC), a widely used approach to deter-mine the number of folding units within a protein molecule Disruption of native PGK structure upon cooling occurs in two distinct stages, corresponding to independent and reversible unfolding of the individual domains [66,70–72] Recently it has been suggested that the uncoupled unfolding of the two domains is a result of the presence of relatively high concentration
of guanidine hydrochloride used in cold denaturation experiments [62] On the other hand, thermal unfolding
of PGK invariably proceeds in an apparently single DSC transition both at low guanidine hydrochloride concentration [69] and in diluted buffer [19,20,67] Strong interdomain stabilization has been claimed in both cases, but the slightly asymmetric thermal unfold-ing profile may be accounted for by assumunfold-ing partially separated unfolding of the N- and C-terminal domains For the protective effect of substrates against thermal unfolding of PGK the experimental data are scarce [19,20,76], no systematic comparison has been made
in the various binary and ternary enzyme–substrate complexes
Separated DSC transitions attributable to the indi-vidual PGK domains have been observed in the case
of engineered mutants of yeast PGK that contain modi-fications in the hinge region between the two domains [20,68] and in the case of cold-active PGK [76], pos-sibly due to weaker interdomain interactions in these cases It is notable that the substrate 3-phospho-d-glycerate (3-PG) in one case [68], while 3-PG and MgADP together in the other case [76] caused merging
of the two transitions into a single one This indicates increased domain cooperativity upon substrate binding
in the mutants Another thermal unfolding study of a substrate-free PGK from the thermophilic bacterium Thermotoga maritima has led to the proposal of a four-state model with three well-defined unfolding transitions: disruptions of domain–domain interactions and subsequent sequential unfolding of the two domains [75]
Domain coupling and dependence on substrate bind-ing are important not only in the stabilizbind-ing mechan-ism of PGK, but also in interdomain communication during the catalytic cycle To characterize the extent of domain coupling and its regulation by each substrate
we have devised thermal unfolding experiments with the mammalian pig muscle PGK, studied in our
Trang 3laboratory For comparison, yeast PGK was also
investigated The purpose was twofold: to resolve, as
much as possible, the overlapping thermal transitions
of the two domains; and to determine the effects of
substrates (in their binary and ternary complexes with
PGK) on the thermal transitions To achieve this, heat
transitions of wild-type pig muscle PGK as well as that
of the wild-type and two single Trp mutants of yeast
PGK were monitored by applying two independent
methods, microcalorimetry and fluorimetry The
try-ptophans of the two mutants are located either in the
N- or in the C-terminal domain [39,41], allowing
selective fluorimetric detection of the conformational
changes within the domains, while DSC calorimetry
characterizes the two domains together Furthermore,
the effects of substrates on thermal unfolding of PGK
are compared in various binary and ternary complexes
and we investigated whether these effects correlate with
the existing molecular interactions, known from the
X-ray structures
Results and Discussion
Interdomain interactions of PGK
Coincidence of calorimetric and fluorimetric heat
transitions of wild-type PGKs reflects domain
co-operativity
To test the extent of domain–domain coupling during
thermal unfolding of PGK, we performed both DSC
and fluorimetric heat transition experiments with the
wild-type pig muscle and yeast PGKs In both
enzymes, the Trp residues, mainly responsible for the
protein fluorescence, are located within the C-terminal
domain: four Trp-s in pig [5], and two Trp-s in the yeast
PGKs [7] Thus, if there is any uncoupling between the
domains, a noncoincidence of the calorimetric and
fluorimetric transition temperatures is expected, even if
their thermal unfolding is not well separated
Typical heat capacity plots of DSC experiments and
fluorimetric thermal transition curves obtained for the
thermal unfolding of the substrate-free pig muscle
PGK and its complexes with various substrates are
shown in Fig 1B and C Part of the curves in Fig 3
illustrates similar experiments with wild-type yeast
PGK The Tmvalues and the experimental calorimetric
heats of unfolding (Qt) are given in Table 1, indicating
pronounced protection by the substrates, details of
which will be discussed later
In all cases the DSC transition curves are
character-ized by single, slightly asymmetric transitions No
residual structure could be detected after the heat
transition by far UV CD spectroscopy (data not
shown) Heat denaturation of pig muscle PGK is an irreversible process, similar to that of the yeast enzyme [19,67] No repeated heat transition was observed on subsequent re-scanning of the sample Furthermore, the observed Tmvalues are found to be strongly scan-rate-dependent, thus, they are at least partially under kinetic control, similar to those of yeast PGK [67] These findings provide evidence of a nonequilibrium unfolding mechanism The irreversible nature of the
50
40
30
20
10
0
40
30
20
10
0 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Temperature (°C)
FN
CP
CP
A
B
C
Fig 1 Effect of substrates on the temperature-dependent unfold-ing of pig muscle PGK DSC (A, B) and fluorimetric (C) heat dena-turation curves were determined at the scanning rate of 1.0 KÆmin)1with carboxamidomethylated (A) and unmodified (B, C) pig muscle PGK in the absence of substrates (d), in the presence
of 10 mM MgATP (n), 10 mM MgADP (m), 10 mM 3-PG (h) and
10 mM MgADP plus 10 mM 3-PG (r) In (A) and (B) the values of excess heat capacity (DC P ) is plotted against temperature.
Trang 4heat transition is also supported by the simultaneous
increase of light scattering during the heat transition
of PGK (data not shown), which indicates occurrence
of accompanying aggregation The mechanism is
poss-ibly a complex one since deviation from the simple
two-state irreversible nativefi unfolded denaturation
model has been claimed from previous DSC
experi-ments with yeast PGK [67] We have also analysed the
present DSC data according to the kinetic model for
irreversible denaturation as described by Sanchez-Ruiz
et al [77] The results in Fig 2A show that, although
the data can be approximated by straight lines, they
indeed, deviate from the simple two-state irreversible
denaturation model, as no common straight line is
formed when different scan rates are applied [78]
Our main purpose, however, was not the
clarifica-tion of the overall mechanism of thermal unfolding
We have restricted ourselves to the question of
whe-ther unfolding of the two domains is (even slightly)
separated Therefore, thermal transitions were also
followed by measuring tryptophan fluorescence
inten-sity changes (Fig 1C) It should be noted that these
transitions are not distorted by the above-mentioned
aggregation, since the extent of aggregation is
propor-tional to the changes in protein fluorescence during the
whole transition Under identical scan rate and
condi-tions, within the experimental error, the same Tm
values were observed in fluorescence (Fig 1C) as in the
DSC-experiments (Fig 1B) and the data are
summar-ized in Table 1 The coincidence of the Tmvalues
deter-mined by fluorimetry and calorimetry indicates that
disruption of the C-domain structure and of the whole
molecule cannot be separated, i.e the two domains are
possibly disrupted in a highly co-operative way
Different heat stabilities of two single Trp mutants
of yeast PGK are not related to domain uncoupling
In the above experiments thermal unfolding of either the whole PGK molecule or its C-terminal domain (within the intact molecule) was monitored In order
to detect unfolding of both N and C domains within the molecule more directly, we performed comparative DSC and fluorimetric heat transition experiments on two single Trp mutants of yeast PGK The Trp residue was either in the N- (W122) or the C-terminal (W333) domain, as described by Mas et al [39,41] Residue numbers 122 and 333 correspond to 123 and 335 in pig muscle PGK, respectively In agreement with previ-ous data [39,41], we found that these mutants are fully active, thus, the mutations do not perturb the structure significantly Stabilities of the mutants are also only slightly decreased with respect to the wild-type enzyme
in guanidine hydrochloride-induced denaturation [41, 62] Previously a sequential domain-unfolding model has been suggested for the mutants According to this model the C domain unfolds first, while the N domain remains relatively compact, but looses most of its ter-tiary structure Complete unfolding of the N domain occurs only during the second transition [41] The reverse order of stabilities, however, has been reported for the isolated N- and C-terminal domains of the mutants [62] Therefore, if the two PGK domains unfold sequentially (in either order) not only in the denaturant-induced, but also in heat denaturation transitions, a different type of noncoincidence of the calorimetric and fluorimetric transition temperatures is expected with the W122 and the W333 mutants, respectively
Table 1 Mid-point temperatures (Tm) and calorimetric heats (Qt) of thermal transitions of pig muscle and yeast PGKs Tmand Qtvalues are given in C and kcalÆmol)1, respectively Tm,caland Tm,fluorwere determined by DSC and fluorimetric experiments, respectively, as shown in Figs 1 and 3 The experimental errors of T m and of Q t were ± 0.2–0.3 C and ± 5–10 kcalÆmol)1, respectively.
Ligand
a Published values, taking into account the decreasing effect of Mg 2+ on PGK stability [83] b Extrapolated to zero concentration of free
Mg 2+ using the method described earlier [83].
Trang 5The most important feature of the results is the
good correlation of the experimental Tm values
(Table 1), determined either calorimetrically (Fig 3A)
or fluorimetrically (Fig 3B) These results strongly
argue in favour of a highly co-operative thermal
unfolding of the two domains in case of both enzyme
forms, although the stabilities of the whole molecules
differ from each other and from that of the wild-type
yeast PGK
Quantitative analysis of the heat transition data, on
one hand, gave straight lines for W333 and wild-type
PGKs (Fig 2B), in agreement with the kinetic model
for irreversible denaturation [77] Non-coincidence of
the calorimetric and fluorescence data, however, indicates deviation from the one-step irreversible nativefi unfolded denaturation model (similar to the findings in Fig 2A) This deviation may influence slightly differently the calorimetric and fluorimetric detection of unfolding For the substrate-free W122 mutant, on the other hand, deviation from the two-state irreversible model is very pronounced This is indicated by both the biphasic nature of the curve in Fig 3B and the well visible deviation from straight lines, especially in case of the more sensitive fluorimet-ric method (Fig 2B) This behaviour of the W122 mutant is consistent with the previously suggested two-step unfolding of the N domain by Mas’ group [40,41]
As this study shows that two-step behaviour is observed for both the calorimetric and fluorimetrically detected heat transitions of the intact molecule, it is conceivable that melting of the two domains, even in this case occurs in a highly co-operative way
The co-operative unfolding mechanism, shown from the experiments with either wild-type or mutant PGKs, differs largely from the sequential mechanism derived previously for refolding of the two domains of pig muscle PGK [60] This is probably due to the fact that) in contrast with refolding ) unfolding starts from the native structure with folded domains and established interdomain interactions, resulting in a stronger coupling between domains
Structural basis of PGK domain cooperativity:
conserved features of the interdomain region
In order to rationalize the structural basis of the highly cooperative domain–domain interactions, indicated by previous and present calorimetric data, we were look-ing for the similarities in 12 available crystal structures
of various PGKs Three different types of molecular contacts were collected and visually investigated: (a) backbone peptide H bonds; (b) electrostatic and H-bonding contacts; (c) hydrophobic interactions between side chains of the conserved residues or between atoms of backbone peptides and of the con-served side chains From these molecular contacts only those that exist in all PGK structures were selected, independently of the source, the conformational state (open or closed) and of ligation with substrates Among the backbone H bonds (Fig 4A) there are special ones (listed in Table 3) which are in crucial positions, directly linking the nearby secondary struc-tural elements to the previously described C- and N-terminal hinges of the interdomain helix 7 [3] as well
as to bL, where the main hinge is possibly located [5] (Fig 4B and C)
-4
-5
-6
-7
-8
-9
-10
3.04
3.00
1.0
0.5
0.0
-0.5
-1.0
-1.5
10 3/T (K-1 )
A
B
Fig 2 Linear transformation of the thermal unfolding data Plots
(A) and (B) were prepared by using Eqns (7) and (8), respectively.
In (A) the data of the DSC transition curves with the substrate free
pig muscle PGK, obtained at scanning rates (v) of 1.5 (n), 0.7 (h),
0.4 (s) and 0.1 () KÆmin)1, were analysed (B) Data of calorimetric
(filled symbols) and fluorimetric (unfilled symbols) measurements,
using the scanning rate of 1.0 KÆmin)1, were compared for
wild-type pig muscle (d,s), yeast (j,h) and W122 (r,e), W333 (m,n)
mutant yeast PGKs in the absence of substrates The original data
are shown in Figs 1B, 1C, 3A and 3B The activation parameters
obtained from plot (A) agreed within the experimental error with
the values given in Table 2.
Trang 6The H bonds at the N-hinge (Fig 4C) create a
con-nection between the C and N terminals of the
polypep-tide chain (a special structural feature of the molecule),
as well as between helices 5 and 7 Here we emphasize
the conservative nature of the H bonds stabilizing this
region and their importance in determining the
posi-tion of the whole N domain relative to the
interdo-main helix 7 A similar role can be attributed to the H
bonds at the C-hinge (Fig 4B) As we show below, in
addition to these permanent bonds, there are further,
changeable H bonds The number and exact location
of these bonds vary upon ligation with the substrates
and with the conformational states of the protein
molecule The changeable H bonds may contribute to
stabilization of the various conformational states,
while the permanent ones may allow a rigid-body-like
movement of the domains relative to helix 7
There are no special H bonds within the
inter-domain region, but the entire region is built up of
conserved residues Their hydrophobic (Fig 4D),
electrostatic and H bonding (Fig 4E) interactions are
listed in Table 3 These contacts of the conserved
resi-dues exist in all PGK structures, independent of their
conformational states (open or closed) or the complex formation with various substrates or ligands An exten-ded hydrophobic cluster dominates in the interdomain interactions The known cold sensitivity of these forces may correlate with the finding that unfolding of the two domains is not coupled during cold denaturation [66,73] This hydrophobic cluster together with the ionic interactions and H bonds constitute a well organ-ized interdomain region which may have great import-ance both in mediating conformational effects between the domains and in unifying the two domains into a single cooperative melting unit at elevated tempera-tures
Stabilization of PGK conformation in the binary substrate complexes
Protection by the individual substrates against thermal unfolding
Both the Tm-values and the experimental calorimetric heats (Qt) required for unfolding (Table 1) are increased significantly by the substrates, indicating their distinct protective effects on PGK conformation
FN
CP
40 35 30
30
25
20
15
10
5
0
1.0
0.8
0.6
0.4
0.2
0.0
A
C
Fig 3 Heat transitions of wild-type and mutant yeast PGKs DSC (A, C) heat denaturation curves of wild-type (j), W122 (r) and W333 (m) yeast PGKs and fluorimetric (B, D) heat denaturation curves of wild-type (h), W122 (e) and W333 (n) yeast PGKs were determined at the scanning rate of 1.0 KÆmin)1, in the absence of substrates (A, B) and in the presence of 10 mM 3-PG (C, D) In (A) and (C) the values of excess heat capacity (DC P ) is plotted against the temperature.
Trang 7(Fig 1) Due to the irreversibility of the folding process,
characterization of the substrate-caused effects
accord-ing to the equilibrium thermodynamics is not possible
The deviation form the two state irreversible model is, however, apparently not too large in most cases (Fig 2) Thus, the reversibly unfolded intermediate
Fig 4 Non-covalent bonds of PGK responsible for interdomain interactions The ribbon diagram of the open conformation of the substrate-free pig muscle PGK [80] (A) and its details at the C-hinge (B), at the N-hinge (C) as well as in the interdomain region including the main hinge at bL (D and E) are shown Various important secondary structure elements are labelled and coloured differently In figures A, B and
C, the backbone of the polypeptide chain is also illustrated (stick model) together with the stabilizing H-bonds (dashed lines) The conserved side-chains (stick models) in the interdomain region are seen with their hydrophobic (D), electrostatic and H-bonding (E) interactions (dashed lines) The interaction distances are listed in Table 3.
Trang 8state(s) may not accumulate in detectable amounts, i.e.
their formation is possibly much slower than their
decay into an irreversibly unfolded state On this basis,
except for the ligand-free W122 mutant of yeast PGK,
we could estimate the kinetic activation parameters of
the process from the type of plot shown in Fig 2A
These parameters are summarized in Table 2 The
sub-strates increase both the activation enthalpy (DH) and
the activation entropy (DS) of unfolding in a way that
finally leads to an increase of the activation free
enthalpy (DG), which quantitatively measures the
sta-bilization effect Of substrates studied 3-PG had the
strongest, MgADP an intermediate and MgATP the
weakest stabilizing effect The observed order of
stabil-ity of the various PGK–substrate complexes is
inter-preted below on structural basis
It is also evident from these results that a similar
cooperative mechanism operates in the binary
com-plexes with either substrate, i.e their stabilizing effect
is not restricted to the N or C domain, respectively, to
which they bind Thus, each of the substrates also
sta-bilizes the domain to which they do not bind,
provi-ding further evidence in favour of operation of strong
domain–domain interactions
Molecular explanation of the increased conformational
stability by 3-PG
The largest protection among the investigated
sub-strates against thermal unfolding of PGK was shown
by 3-PG, which is in agreement with spectroscopic
studies [29]
To describe the effect of substrates in structural
terms, we have searched for new atomic contacts
within the protein formed only upon substrate binding
The contacts between the bound 3-PG and PGK,
known from crystallographic studies of this binary
complex of pig muscle PGK [8] (Fig 5A) as well as from other 3-PG containing PGK structures [3–5,79– 81] suggest a possible way of stabilization by 3-PG Namely, all side chains interacting with 3-PG belong
to separate structural elements of the N domain, namely bA, bB, bD and bE as well as helices 1 and 5 Thus, these structural elements are strongly fixed together by 3-PG and this may result in an increased stability of the whole N domain
Calorimetric experiments, however, indicate that 3-PG and other substrates stabilize the whole mole-cule, not only the domain to which they bind This effect is most probably promoted by the existing inter-actions between the two domains The transmission of 3-PG induced effects from the N domain to the C domain may be visualized by observing the newly formed interactions within the protein molecule (col-oured violet in Fig 5A, Table 3), characteristic of all 3-PG-bound PGK structures The Arg38 side chain (helix 1) makes new H bonding with Thr393 (peptide
O atom) in bL, and by the aid of the permanent elec-trostatic interaction with the carboxylate of Asp23 (bA) a new connection is formed between the two domains Numbering of residues throughout the text refers to pig muscle PGK sequences, unless stated otherwise e.g Bacillus stearothermophilus (Bs) or Try-panosoma brucei (Tb) This connection is characteristic
of all 3-PG-bound structures [3,4,81,82], whereas bind-ing of MgADP or MgATP to the substrate-free enzyme does not induce formation of this bond (Table 3)
Based on structural comparison we present here a possible mechanism of the conformational changes caused by 3-PG that lead to the formation of the Arg38–Thr393 interaction Upon 3-PG binding the strong interaction between its phosphate and the side chain of Arg170 on helix 5 shifts the whole helix 5,
Table 2 The activation parameters of thermal transitions of pig muscle and yeast PGKs The values of DH (kcalÆmol)1) and DS (calÆmol)1) were derived from the type of plot shown in Fig 2A, prepared from DSC measurements DH was assumed to be independent of the tem-perature within the range of the measurements The calculated DG (kcalÆmol)1) values are referred to 25 C The errors of DH and DS are
± 10–15%.
Ligand
Trang 9thereby the ring of Phe165 (see Fig 4D) is also
dis-placed parallel to its former position by at least about
2 A˚ This effect is further enhanced through the
inter-action with Glu192 (helix 7) and causes an additional
shift of about 3.5 A˚ in the position of the imidazole ring
of the interacting His390 (Fig 4E) Since His390 is
located in bL, the conformation of this b-strand may
also become significantly altered This conformational
change would be directly related to the domain
move-ment, since there are strong arguments supporting that
the main molecular hinge of PGK is located in bL [5]
This may be the explanation for the small extent of
domain rotation observed in the 3-PG binary complex
[8] The new conformation of bL is stabilized by a new
H bond between the conserved Ser392(OG) and
Gly394(N) (Fig 5A), characteristic of all PGK
struc-tures which bind 3-PG These changes also lead to a
roughly 2 A˚ shift of the backbone atoms of Thr393
towards the guanidium group of Arg38 and the two
may reach each other within H-bonding distance The
conformational change caused by 3-PG binding in bL
can be further transmitted to the C domain through the
H-bonding system between bL and bK shown in
Fig 4B During this process formation of new H
bonds between Thr375(OG1) (from helix 13 that is
sequentially situated between bK and bL) and
Gly337(N), as well as between the nearby Val339(N)
and Thr351(OG1) (belonging to helix 12) may
streng-then the interactions within the C domain (Fig 5A) It
is notable, that Gly337 and Val339 are in a loop
between bJ and helix 12, which is just below the binding
site of the nucleotide substrate These latter H bonds
also exist in the binary complex with the nucleotide
sub-strates, but they are absent in the substrate-free PGK
Through the contacts described above, the conform-ational changes induced by substrate binding can be transmitted from the 3-PG-site of the N domain to the nucleotide binding pocket of the C domain leading to stabilization the whole enzyme molecule
Fig 5 Details of the interdomain region in the binary complexes
with substrates (A) and (C) show 3-PG and MgATP binding,
respectively, to pig muscle PGK [8,83], while MgADP binding to
B stearothermophilus PGK [9] is shown in (B) In each case
sequence numbering refers to the corresponding species The
important secondary structural elements are highlighted as ribbons
with the same colour as shown in Fig 4 Blue ball and stick models
represent the bound substrates Only the side-chain or backbone
atoms (stick models) interacting with the substrates and the ones
forming new interactions in the protein molecule, characteristic of
the substrate-bound structures are shown and coloured violet The
interacting atoms are connected with dashed lines, while arrows
connect the equivalent noninteracting atoms in C In the latter
case, the distances are also indicated in angstroms One
perma-nent peptide H bond (370:O )392:N in A or Bs348:O–Bs370:N in B)
that makes connection between bK (green) and bL (red),
character-istic of all PGK structures, is also indicated (dashed lines) The
protein contacts together with the distances between the
corres-ponding atoms in the substrate-free and the closed ternary
com-plex structures are listed in Table 3.
Trang 10Table 3 Atomic contacts responsible for domain cooperation Atomic distances (A ˚ -s) were measured in the interdomain region and its sur-roundings From the total of 12 crystal structures investigated, data of those most characteristic were selected The contacts that vary upon substrate binding or upon domain closure are indicated in bold The contact list for the closed structure of T maritima PGK (data not shown)
is similar to that of T brucei PGK, with few exceptions.
Interacting
structural
elements
Pig muscle PGK (open) B stearotherm PGK (open) T brucei PGK (closed)
Atom 1 Atom 2
Substrate free
3-PG binary MgATP binary Atom 1 Atom 2
MgADP binary Atom 1 Atom 2 Ternaryc Backbone H bonds
Within helix 7
(C-terminal part)
Helix 7 (C-term.) and bJ E204:Oa N332:N 4.44 4.14 4.50 D186:O L312:N 4.16 P208:O K334:N 3.27
Hydrophobic interactions
Helix 5 and helix 7 F165:CG E192:CG 3.64 3.58 3.69 F146:CG E174:CG 3.86 F167:CG E196:CG 3.53
F165:CD2 E192:CB 3.68 3.54 3.74 F146:CD2 E174:CB 3.85 F167:CD2 E196:CB 3.38 F165:CE1 E192:CD 3.72 3.85 4.08 F146:CE1 E174:CD 4.47 F167:CE2 E196:CD 3.72 F165:CZ F196:CE1 3.73 4.19 4.15 F146:CZ L178:CD1 3.72 F167:CZ F200:CE1 3.36 Helix 5 and helix 14 F165:CD1 G394:CA 3.98 3.64 4.06 F146:CD1 G372:CA 3.65 F167:CD2 G397:CA 4.01
F165:CZ L401:CD1 4.18 3.79 4.08 F146:CZ F379:CD2 4.38 F167:CZ L404:CD2 3.95
Helix 7 and C-terminus L193:CD1 V410:CG1 4.35 4.35 4.54 L175:CD1 V388:CG1 4.11 I197:CD1 V413:CG1 3.55 Side-chain H bonds and electrostatic interactions
Loop after bB and helix 8 R65:NH2a D218:OD2 16.25 12.58 14.89 R62:OD1 D200:NH1 16.54 R65:NH1 D222:OD1 3.14
bE and helix 7 (N-term.) D163:OD1 L188:N 2.96 2.88 2.86 D144:OD2 L170:N 2.88 D165:OD1 L192:N 2.84
Helix 5 and helix 14 H169:ND1 E400:OE2 3.76 3.77 4.50 H150:ND1 E378:OE2 3.74 H171:ND1 E403:OE1 3.20 Helix 7 and bL E192:OE1 H390:NE2 2.99 2.89 2.82 E174:OE1 H368:NE2 2.90 E196:OE1 H393:NE2 3.06
E192:OE1 S392:OG 2.74 2.92 2.80 E174:OE1 S370:OG 2.72 E196:OE1 S395:OG 2.64
E192:OE2 T393:OG1 2.75 2.95 2.56 E174:OE2 T371:OG1 2.79 E196:OE2 T396:OG1 2.48 Helix 8 and bJ K219:NZ b N336:OD1 4.65 6.64 6.77 K201:NZ N316:OD1 2.81 K223:NZ N338:OD1 3.23