Báo cáo khoa học: Correlation between conformational stability of the ternary enzyme–substrate complex and domain closure of 3-phosphoglycerate kinase potx

Correlation between conformational stability of the ternary enzyme–substrate complex and domain closure of 3-phosphoglycerate kinase ´ ´ ´ ´ ´ Andrea Varga1, Beata Flachner1, Eva Graczer1, Szabolcs Osvath2, Andrea N Szilagyi1 ´ and Maria Vas1 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary 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 hydrophobic 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 The experimental and theoretical studies that led to our present understanding of protein structural changes 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 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 FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS 1867 Substrate-assisted domain–domain cooperativity this 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 degradation [16,17] and unfolding [12,18–20] has been observed Substrate-induced conformational changes have been detected by various techniques, including NMR [21–23], fluorescence [15,24–29] and ESRspectroscopy [30], analytical ultracentrifugation [31], small angle X-ray scattering [32–34] and X-ray crystallography [3,4,8] There are, however, conflicting results concerning the requirement of either only one or both substrates in causing domain movement and the question 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 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 closure by the lattice forces operating in certain PGK crystals [35] The best approach by which to obtain direct information about the extent of domain–domain cooperativity, is to carry out unfolding–refolding experiments, as noninteracting structural domains generally correspond to separate folding units Both unfolding [36–50] and refolding [36–38,46–62] experiments using denaturants 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 unfolding ⁄ refolding of the two domains probably occurs in a sequential order within the PGK molecule No comprehensive picture, however, has yet emerged about the relative stability of the two domains and about the 1868 A Varga et al 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 determine 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 unfolding profile may be accounted for by assuming 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 individual PGK domains have been observed in the case of engineered mutants of yeast PGK that contain modifications in the hinge region between the two domains [20,68] and in the case of cold-active PGK [76], possibly due to weaker interdomain interactions in these cases It is notable that the substrate 3-phospho-dglycerate (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 binding are important not only in the stabilizing mechanism 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 FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS A Varga et al 50 A ∆CP (kcal/mole/°C) 40 B 40 FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS 30 20 10 1.2 C Interdomain interactions of PGK 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 illustrates similar experiments with wild-type yeast PGK The Tm values 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 characterized by single, slightly asymmetric transitions No residual structure could be detected after the heat transition by far UV CD spectroscopy (data not 20 Results and Discussion 1.0 0.8 FN Coincidence of calorimetric and fluorimetric heat transitions of wild-type PGKs reflects domain co-operativity 30 10 ∆CP (kcal/mole/°C) laboratory 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 tryptophans 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 Substrate-assisted domain–domain cooperativity 0.6 0.4 0.2 0.0 40 45 50 55 60 65 Temperature (°C) Fig Effect of substrates on the temperature-dependent unfolding of pig muscle PGK DSC (A, B) and fluorimetric (C) heat denaturation curves were determined at the scanning rate of 1.0 KỈmin)1 with 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 (DCP) is plotted against temperature 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 Tm values are found to be strongly scanrate-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 1869 Substrate-assisted domain–domain cooperativity A Varga et al Table Mid-point temperatures (Tm) and calorimetric heats (Qt) of thermal transitions of pig muscle and yeast PGKs Tm and Qt values are given in °C and kcalỈmol)1, respectively Tm,cal and Tm,fluor were determined by DSC and fluorimetric experiments, respectively, as shown in Figs and The experimental errors of Tm and of Qt were ± 0.2–0.3 °C and ± 5–10 kcalỈmol)1, respectively Pig muscle PGK Yeast PGK Unmodified CM- Ligand Tm,cal Tm,fluor Qt Tm,cal No MgATP 53.0 54.8 55.6a 56.9 57.6a 58.2 59.1 59.8b 53.1 55.3 – 57.0 – 57.5 58.6 – 114 125 – 139 – 147 175 – 46.5 47.6 – 48.7 – 52.4 51.1 – MgADP 3-PG 3-PG + MgADP Wild-type Qt 85 91 – 98 – 110 105 – W122 Tm,cal Tm,fluor Qt Tm,cal Tm,fluor Qt Tm,cal Tm,fluor Qt 56.4 59.2 – 60.7 – 60.1 62.8 – 56.2 59.8 – 61.4 – 60.0 63.4 – 110 127 – 140 – 150 157 – 47.5 49.4 – 51.5 – 51.4 53.7 – 48.5 51.3 – 52.3 – 51.7 54.0 – 49 60 – 73 – 79 89 – 52.6 56.0 – 57.0 – 56.6 58.9 – 52.9 55.7 – 57.5 – 57.2 59.6 – 103 126 – 130 – 131 150 – a Published values, taking into account the decreasing effect of Mg2+ on PGK stability [83] Mg2+ using the method described earlier [83] heat 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 possibly a complex one since deviation from the simple two-state irreversible native fi unfolded denaturation model has been claimed from previous DSC experiments 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 clarification of the overall mechanism of thermal unfolding We have restricted ourselves to the question of whether unfolding of the two domains is (even slightly) separated Therefore, thermal transitions were also followed by measuring tryptophan fluorescence intensity changes (Fig 1C) It should be noted that these transitions are not distorted by the above-mentioned aggregation, since the extent of aggregation is proportional to the changes in protein fluorescence during the whole transition Under identical scan rate and conditions, 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 summarized in Table The coincidence of the Tm values determined 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 1870 W333 b Extrapolated to zero concentration of free 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 previous data [39,41], we found that these mutants are fully active, thus, the mutations 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 tertiary 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 FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS A Varga et al Substrate-assisted domain–domain cooperativity -4 A -5 In(k/T) -6 -7 -8 -9 -10 3.04 3.06 3.08 3.10 3.12 1.0 In(In[N]0/[N]) or In(In(Qt/(Qt-Q))) B 0.5 0.0 -0.5 -1.0 -1.5 3.00 3.05 3.10 3.15 3.20 103/T (K-1) Fig 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 (Đ) KỈmin)1, were analysed (B) Data of calorimetric (filled symbols) and fluorimetric (unfilled symbols) measurements, using the scanning rate of 1.0 KỈmin)1, were compared for wildtype 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 The 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 FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS the calorimetric and fluorescence data, however, indicates deviation from the one-step irreversible native fi 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 twostate 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 fluorimetric method (Fig 2B) This behaviour of the W122 mutant is consistent with the previously suggested twostep 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 looking 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 conserved 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 structural elements to the previously described C- and N-terminal hinges of the interdomain helix [3] as well as to bL, where the main hinge is possibly located [5] (Fig 4B and C) 1871 Substrate-assisted domain–domain cooperativity A Varga et al 30 A C B D 25 ∆CP (kcal/mole/°C) 20 15 10 1.0 0.8 FN 0.6 0.4 0.2 0.0 30 35 40 45 50 55 60 Temperature (°C) 35 40 45 50 55 60 65 Temperature (°C) Fig 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 KỈmin)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 (DCP) is plotted against the temperature The H bonds at the N-hinge (Fig 4C) create a connection between the C and N terminals of the polypeptide chain (a special structural feature of the molecule), as well as between helices and Here we emphasize the conservative nature of the H bonds stabilizing this region and their importance in determining the position of the whole N domain relative to the interdomain helix 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 There are no special H bonds within the interdomain 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 These contacts of the conserved residues exist in all PGK structures, independent of their 1872 conformational states (open or closed) or the complex formation with various substrates or ligands An extended 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 organized interdomain region which may have great importance both in mediating conformational effects between the domains and in unifying the two domains into a single cooperative melting unit at elevated temperatures 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 FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS A Varga et al Substrate-assisted domain–domain cooperativity Fig Non-covalent bonds of PGK responsible for interdomain interactions The ribbon diagram of the open conformation of the substratefree 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 (Fig 1) Due to the irreversibility of the folding process, characterization of the substrate-caused effects according to the equilibrium thermodynamics is not possible FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS The deviation form the two state irreversible model is, however, apparently not too large in most cases (Fig 2) Thus, the reversibly unfolded intermediate 1873 Substrate-assisted domain–domain cooperativity A Varga et al Table The activation parameters of thermal transitions of pig muscle and yeast PGKs The values of DHà (kcalỈmol)1) and DSà (calỈmol)1) were derived from the type of plot shown in Fig 2A, prepared from DSC measurements DHà was assumed to be independent of the temperature within the range of the measurements The calculated DGà (kcalỈmol)1) values are referred to 25 °C The errors of DHà and DSà are ± 10–15% Pig muscle PGK Yeast PGK Unmodified CM- Wild type W122 W333 Ligand DHà DSà DGà DHà DSà DGà DHà DSà DGà DHà DSà DGà DHà DSà DGà No MgATP MgADP 3-PG 3-PG + MgADP 129 160 204 174 241 329 418 551 457 658 31.0 35.1 39.6 37.3 44.9 128 137 127 122 132 333 359 328 307 339 28.9 29.9 29.4 30.6 30.8 124 131 149 199 247 310 326 380 530 671 32.0 33.5 35.8 40.8 47.5 – 120 138 161 174 – 303 357 428 465 – 29.1 31.3 33.4 35.3 165 197 190 224 242 438 530 509 612 663 33.9 38.5 38.2 41.6 44.7 state(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 The substrates increase both the activation enthalpy (DHà) and the activation entropy (DSà) of unfolding in a way that finally leads to an increase of the activation free enthalpy (DGà), which quantitatively measures the stabilization effect Of substrates studied 3-PG had the strongest, MgADP an intermediate and MgATP the weakest stabilizing effect The observed order of stability of the various PGK–substrate complexes is interpreted below on structural basis It is also evident from these results that a similar cooperative mechanism operates in the binary complexes 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 stabilizes the domain to which they not bind, providing 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 substrates 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 1874 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 and 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 molecule, not only the domain to which they bind This effect is most probably promoted by the existing interactions 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 (coloured 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 electrostatic 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 Trypanosoma brucei (Tb) This connection is characteristic of all 3-PG-bound structures [3,4,81,82], whereas binding 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 shifts the whole helix 5, FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS A Varga et al Substrate-assisted domain–domain cooperativity Fig 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 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 permanent 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), characteristic of all PGK structures, is also indicated (dashed lines) The protein contacts together with the distances between the corresponding atoms in the substrate-free and the closed ternary complex structures are listed in Table thereby the ring of Phe165 (see Fig 4D) is also displaced parallel to its former position by at least about ˚ A This effect is further enhanced through the interaction 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 movement, 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 structures which bind 3-PG These changes also lead to a ˚ roughly 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 strengthen 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 substrates, but they are absent in the substrate-free PGK FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS Through the contacts described above, the conformational 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 1875 Substrate-assisted domain–domain cooperativity A Varga et al ˚ Table Atomic contacts responsible for domain cooperation Atomic distances (A-s) were measured in the interdomain region and its surroundings 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 Pig muscle PGK (open) Interacting structural elements Backbone H bonds N- and C-terminals N-terminus and bF Helix and C terminus bF and C terminus Within helix (C-terminal part) Helix (C-term.) and bJ bG and bH bG and bJ bK and bL Within helix 13 B stearotherm PGK (open) Substrate 3-PG MgATP free binary binary Atom Atom Ternaryc Q5:O K7:N K7:O I9:N A170:O G187:O V203:O V203:O N206:O P207:O P208:O L211:O A213:O P210:O P210:O V212:N I214:O I373:O G375:N G375:N A382:N Q383:N K419:N D417:O A189:N A189:O G412:N D417:N G205:N P207:N P208:N R209:N K334:N D235:N L238:N K333:N S334:N S334:O N338:N S395:N S395:O G397:O S378:O A379:O 2.84 2.79 2.84 3.22 2.72 2.90 3.22 3.32 3.41 3.25 3.27 2.73 2.82 2.83 3.04 2.97 2.85 2.70 3.46 3.24 2.96 2.96 3.86 3.85 4.47 3.72 3.65 4.69 4.16 4.38 3.74 4.11 F167:CG F167:CD2 F167:CE2 F167:CZ F167:CD2 F167:CZ F167:CZ F167:CZ E196:CD I197:CD1 E196:CG E196:CB E196:CD F200:CE1 G397:CA A400:CB S401:CA L404:CD2 H393:CE1 V413:CG1 3.53 3.38 3.72 3.36 4.01 4.41 4.40 3.95 4.39 3.55 R36:NE2 4.01 T371:O 5.57 T371:O 4.76 D144:OD1 2.64 F169:N 2.97 D200:NH1 16.54 L170:N 2.88 M171:N 3.00 E378:OE2 3.74 H368:NE2 2.90 S370:OG 2.72 T371:N 2.95 T371:OG1 2.79 N316:OD1 2.81 D24:OD2 R39:NE R39:NH2 T46:OG1 T46:OG1 R65:NH1 D165:OD1 D165:OD1 H171:ND1 E196:OE1 E196:OE1 E196:OE2 E196:OE2 K223:NZ R39:NE T396:O T396:O D165:OD2 Y191:N D222:OD1 L192:N M193:N E403:OE1 H393:NE2 S395:OG T396:N T396:OG1 N338:OD1 3.38 2.88 2.93 2.73 2.87 3.14 2.84 2.88 3.20 3.06 2.64 2.86 2.48 3.23 Atom N4:O L6:N L6:O L8:N A168:O K183:O A199:Oa A199:Oa S202:Oa P203:Oa E204:Oa F207:O A209:O P206:O P206:O L208:N I210:O I370:O G372:Na G372:Na C379:Nb A380:Nb V416:N S414:O G185:N G185:O G409:N S414:N E201:N P203:N E204:N R205:N N332:N N231:N I234:N K331:N Q332:N Q332:O N336:N S392:N S392:O G394:O T375:O A376:O 3.00 2.74 2.98 2.66 2.83 2.68 4.42 5.54 4.03 3.80 4.44 2.83 2.84 2.71 3.25 2.89 2.95 2.97 4.62 4.47 3.82 3.88 2.98 2.78 2.78 2.92 2.81 2.98 3.60 4.19 3.89 3.75 4.14 2.71 2.91 2.85 3.35 2.89 2.92 2.90 4.16 4.90 3.20 3.45 2.46 2.66 2.85 2.94 2.69 2.83 4.37 5.67 4.08 4.13 4.50 2.98 2.87 2.88 3.14 2.83 2.94 2.97 4.15 3.98 3.41 3.73 N2:O K4:N K4:O I6:N A149:O A165:O A181:O A181:O N184:O P185:O D186:O F189:O A191:O P188:O P188:O T190:N I192:O I348:O G350:N G350:N V357:N E358:N K394:N Q392:O A167:N A167:O G387:N E392:N S183:N P185:N D186:N R187:N L312:N D213:N I216:N K311:N L312:N L312:O N316:N S370:N S370:O G372:O S353:O A354:O 2.74 2.56 3.00 3.22 2.76 2.96 3.89 4.07 3.74 3.77 4.16 2.79 2.86 2.82 3.37 2.99 2.96 2.90 5.36 7.20 3.18 3.05 E192:CG E192:CB E192:CD F196:CE1 G394:CA A397:CB S398:CA L401:CD1 H390:CE1 V410:CG1 3.64 3.68 3.72 3.73 3.98 4.06 3.91 4.18 3.59 4.35 3.58 3.54 3.85 4.19 3.64 4.35 4.50 3.79 3.83 4.35 3.69 3.74 4.08 4.15 4.06 4.36 4.07 4.08 3.50 4.54 F146:CG F146:CD2 F146:CE1 F146:CZ F146:CD1 F146:CZ F146:CZ F146:CZ E174:CD L175:CD1 E174:CG E174:CB E174:CD L178:CD1 G372:CA A375:CB S376:CA F379:CD2 H368:CE1 V388:CG1 3.23 2.80 4.04 5.95 2.68 5.17 2.67 2.60 2.98 2.92 12.58 14.89 2.88 2.86 2.87 2.89 3.77 4.50 2.89 2.82 2.92 2.80 3.03 2.69 2.95 2.56 6.64 6.77 D21:OD2 R36:NE R36:NH2 T43:OG1 T43:OG1 R62:OD1 D144:OD2 D144:OD2 H150:ND1 E174:OE1 E174:OE1 E174:OE2 E174:OE2 K201:NZ Side-chain H bonds and electrostatic interactions bA and helix D23:OD2 R38:NE 2.99 Helix and bL R38:NEa T393:O 5.81 R38:NH2b T393:O 5.16 Helix and bE S45:OG D163:OD2 2.51 Helix and bF S45:OG F187:N 2.88 Loop after bB and helix R65:NH2a D218:OD2 16.25 bE and helix (N-term.) D163:OD1 L188:N 2.96 D163:OD1 M189:N 3.04 Helix and helix 14 H169:ND1 E400:OE2 3.76 Helix and bL E192:OE1 H390:NE2 2.99 E192:OE1 S392:OG 2.74 E192:OE2 T393:N 2.63 E192:OE2 T393:OG1 2.75 Helix and bJ K219:NZb N336:OD1 4.65 1876 MgADP binary Atom Atom Hydrophobic interactions Helix and helix F165:CG F165:CD2 F165:CE1 F165:CZ Helix and helix 14 F165:CD1 F165:CZ F165:CZ F165:CZ Helix and bL E192:CD Helix and C-terminus L193:CD1 T brucei PGK (closed) Atom FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS A Varga et al Substrate-assisted domain–domain cooperativity Table (Continued) Pig muscle PGK (open) Interacting structural elements Helix and helix 14 Atom N225:ND2 N225:ND2a bJ and bK N336:ND2b bJ and helix 13 G337:Nb Helix 12 and loop after bJ T351:OG1b bK and helix 13 G371:Oa Within bL S392:OG S392:OGb G394:N bL and helix 14 S392:OGb a c B stearotherm PGK (open) T brucei PGK (closed) Atom Substrate 3-PG MgATP free binary binary Atom Atom MgADP binary Atom Atom Ternaryc L402:O E403:OE1 G371:O T375:OG1 V339:N T375:OG1 T393:N G394:N T393:OG1 S398:OG 3.44 8.92 6.55 4.75 4.15 3.56 3.51 4.03 3.30 4.38 M380:O E381:OE1 G349:O S353:OG M319:N S353:OG T371:N G372:N T371:OG1 S376:OG 2.99 9.08 3.04 2.67 2.86 3.68 3.09 3.42 3.31 2.72 L405:O E406:OE1 G374:O S378:OG M341:N S378:OG T396:N G397:N T396:OG1 S401:OG 6.31 3.51 2.72 2.93 3.00 3.03 3.01 2.84 3.42 3.00 2.70 8.77 4.21 3.34 3.18 5.42 3.10 2.97 3.37 4.58 2.71 8.64 4.90 3.03 3.39 3.59 3.26 3.58 3.52 4.26 Contacts formed only in the closed structure of T brucei PGK [79]; MgADP*3-PG ternary complex Different stabilizing effects of MgATP and MgADP are due to their different binding Different effects of the two nucleotides can be rationalized by their different binding modes to PGK, suggested from thiol-reactivity studies [13], phosphorescence life-time measurements [28] and supported by X-ray crystallography [9,83] Here we present further insight into these differences Two crystal structures have been published with bound MgADP: a binary complex of the bacterial B stearothermophilus PGK [9], and a ternary complex of T brucei PGK containing bound MgADP and 3-PG [79] In spite of their different natures, the molecular details of MgADP binding are very similar in these complexes Only one binary complex crystal structure exists with bound MgATP [83], but there are several structures with bound MgATP analogues [4,80,81,84] The locations and interactions of the adenine and ribose rings in the C domain are very similar in all cases The occupation and interactions of the nucleotide phosphate chain, however, are strikingly different, in agreement with solution binding data [83] Fluctuation of the flexible phosphate chain of MgATP between two alternative sites was assumed, while no such argument was required by the wellpositioned phosphates of MgADP [83] The existence of two alternative sites for the phosphates of the trinucleotide has, indeed, been shown by the crystallographic studies with different MgATP analogues [4,80,81,84] The binding of MgADP and MgATP are illustrated by the structures of B stearothermophilus [9] and pig muscle [83] PGKs, respectively, in Fig 5B and C The common characteristics of their binding are H-bonding FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS N207:ND2 N207:ND2 N316:ND2 G317:N T331:OG1 G349:O S370:OG S370:OG G372:N S370:OG b N229:ND2 N229:ND2 N338:ND2 G339:N T353:OG1 G374:O S395:OG S395:OG G397:N S395:OG Contacts formed only in the presence of either substrate and hydrophobic interactions of the adenine ring to bq, and of the ribose ring to the loop between bJ and helix 12, as well as the ionic interaction of the a-phosphate with Lys Bs201 ⁄ 219(NZ) in Bs ⁄ pig muscle PGK (Fig 5B and C) at the N terminus of helix Thus, these secondary structural elements may be similarly fixed together in the C domain either by MgADP or MgATP There are, however, further interactions, that are different for the two nucleotides In contrast to MgATP [83], for MgADP [9] both the a- and b-phosphates are linked through the bound Mg2+ to the carboxylate of an aspartate residue (Asp Bs352 ⁄ 374 in Fig 5B; the second number refers to pig muscle PGK) located in helix 13 The b-phosphate of MgADP is linked to Ser Bs353 ⁄ Thr375(OG) from the same helix (Fig 5B) These multiple interactions of MgADP with helix 13 are further strengthened by H bonds and electrostatic forces between its positively charged N terminus and the b-phosphate of the nucleotide The above interactions result in complete ordering of the helix as observed by the X-ray data [9] In addition, a b-phosphate O-atom of MgADP is H bonded to the ND2 atom of Asn Bs316 ⁄ 336 from b-strand J (Fig 5B) Neither of these interactions is formed with MgATP (Fig 5C), therefore helix 13 is not completely ordered in this binary complex structure In addition to the observations noted in the crystallographic papers, here we point out important differences in formation of contacts between conserved side chains In the structure of B stearothermophilus PGK with bound MgADP (Fig 5B) Asn Bs316 ⁄ 336 makes new H bonds These are formed between Asn Bs316 ⁄ 336(OD1) and Lys Bs201 ⁄ 219(NZ) as well as between Asn Bs316 ⁄ 336(ND2) and Gly Bs349 ⁄ 371(O) 1877 Substrate-assisted domain–domain cooperativity (Fig 5B) As indicated by the large atomic distances in Table 3, no such interactions exist in the binary complex with MgATP (Fig 5C) Thus, Asn Bs316 ⁄ 336 from b-strand J has as many as three interactions including the one with b-phosphate of MgADP and thereby it connects bJ with the N terminus of helix (Lys Bs201 ⁄ 219) and with the loop between bK and the N terminus of helix 13 (Gly Bs349 ⁄ 371) These bonds are characteristic only to the MgADP complexes [3,9] and are most probably responsible for the larger conformational stability of PGK in the presence of MgADP compared to that of the MgATP complex, as indicated by our calorimetric measurements The interactions above are not restricted to the C domain Although it is not seen how the effect of the nucleotide binding can reach the N domain, the permanent contacts of the interdomain region assure transmission of its effect In fact, there is a notable new contact of Ser Bs370 ⁄ 392(OG) of bL with Bs376 ⁄ 398(OG) at the beginning of helix 14 (belonging to the N domain) in the presence of MgADP (Fig 5B) This contact does not exist in the MgATP complex (Fig 5C and Table 3), the phosphate chain of which occupies an intermediate position between its two possible alternative sites [83] These observations are in line with the cooperative unfolding of the two domains in the binary complexes with the nucleotides and with the smaller protecting effect of MgATP than MgADP Highest stability of the active ternary complex is due to domain closure Simultaneous protection by the two substrates against thermal unfolding The thermal stability of PGK was further increased in the ternary complexes, i.e in the simultaneous presence of two substrates (Fig 1B and C and Table 1) In order to avoid the heat effect due to the enzyme reaction, an unproductive ternary complex was investigated in which instead of the substrate, MgATP, the product, MgADP was present After analysing the data, similarly to the binary complexes, the activation parameters (Table 2) were estimated These correlate well with the increased stability of the ternary complex vs the binary ones This increased stability of the ternary complex may simply be due to simultaneous formation of the characteristic contacts formed in the presence of the individual substrates Thus, even without further conformational changes (such as domain closure), the stabilizing effects of the two bound substrates may be 1878 A Varga et al additive There are, however, convincing arguments and experimental evidence in favour of PGK domain closure occurring concomitantly with the enzyme– substrate complex formation Since from the above calorimetric experiments one cannot distinguish between the conformational stabilities of the open and closed conformations, in the further work we followed two different strategies: (a) by analysing the atomic coordinates of two different (open and closed) PGK crystal structures we searched for formation of additional atomic interactions that may be responsible for stabilization of the closed conformation over the open one; (b) we devised further calorimetric experiments with a modified PGK that lacks the ability of domain closure Structural origin of the increased stability of the closed conformation In the structures of the two closed ternary complexes of Thermotoga maritima [4] and T brucei PGK [3] the characteristic contacts formed upon the separate binding of 3-PG or MgADP co-exist The main features of the contact pattern are similar in the two closed crystal structures and therefore we discuss only that of the fully closed T brucei PGK (Fig 6) In detail, on the one hand, the bonds between Arg Tb39 ⁄ Bs36 ⁄ 38(NH2) and Thr Tb396 ⁄ Bs371 ⁄ 393(O) (the last numbering refers to pig muscle PGK) as well as between Gly Tb397 ⁄ Bs372 ⁄ 394(N) and Ser Tb395 ⁄ Bs370 ⁄ 392(OG), characteristic of the 3-PG binary complex, also exist in the closed structure of T brucei PGK On the other hand, the bond between Ser Tb395 ⁄ Bs370 ⁄ 392(OG) and Ser Tb401 ⁄ Bs376 ⁄ 398(OG) as well as the double interactions of Asn Tb338 ⁄ Bs316 ⁄ 336 with Lys Tb223 ⁄ Bs201 ⁄ 219(NZ) and with Gly Tb374 ⁄ Bs349 ⁄ 371(O), characteristic of the MgADP binary complex, are also formed in the closed ternary complex (Fig and Table 3) Additional atomic contacts (coloured violet in Fig 6), however, are also present in the closed ternary complex structure of T brucei PGK compared to those existing separately in the two binary complexes The contacts that are different in the closed ternary and the open binary complexes are highlighted in Table The contact between Arg Tb39 ⁄ Bs36 ⁄ 38(NE) and Thr Tb396 ⁄ Bs371 ⁄ 393(O) is essentially characteristic of the closed conformation It further strengthens the contact of the Arg side chain with bL, already formed in the binary complex with 3-PG The formation of a new H bond in the closed structure of T brucei PGK, immediately before helix 13, that connects Gly Tb374 ⁄ Bs349 ⁄ 371(O) to Ser ⁄ Thr Tb378 ⁄ FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS A Varga et al Substrate-assisted domain–domain cooperativity Fig Details of the molecular contacts stabilizing the closed conformation The Ca trace (black) of the molecule of T brucei [79], PGK is shown together with the blue ball and stick models of 3-PG and MgADP The Ca traces of the important secondary structural elements are coloured in the same way as in Figs and From the side-chain and backbone atoms (stick models) only those are shown (in the colour of the corresponding secondary structural elements) that either directly interact with the substrates or are involved in important contacts within the protein and exist already in the respective binary substrate complexes The side-chain or backbone atoms that participate in newly formed contacts in the closed structure are coloured violet The interacting atoms are connected with dashed lines The contacts within the protein together with the distances between the corresponding atoms are listed in Table Bs353 ⁄ 375(OG), stabilizes a new conformation of the N terminus of helix 13 Another peptide H bond between the atoms of Gly Tb375 ⁄ Bs350 ⁄ 372(N) and Ser Tb395 ⁄ Bs370 ⁄ 392(O) (as well as between Gly Tb375 ⁄ Bs350 ⁄ 372(N) and Gly Tb397 ⁄ Bs352 ⁄ 374(O)) of the closed structure creates a new contact between bK and bL, i.e the b strands before and after helix 13 in the sequence As a further consequence of domain closure, a new salt bridge is also formed between Arg Tb65 ⁄ Bs62 ⁄ 65 (N domain) and Asp Tb222 ⁄ Bs200 ⁄ 218 (C domain) that keep the two domains together (Fig 6), while the equivalent residues in the open structure are far away (Table 3) These additional contacts are, in fact, characteristic of the closed conformation, since almost all of them are absent in the open conformation of crystalline pig muscle PGK ternary complex [81], where domain closure was shown to be prevented by the crystal lattice forces [35] The increased number of molecular contacts in the interdomain region of the closed ternary complex, compared to the open binary ones, suggests a larger increase of conformational stability than could have FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS been expected on the basis of simple additive effects of the two simultaneously bound substrates Protecting effect of substrates against thermal unfolding is not additive for carboxamidomethylated (CM)-PGK In order to test experimentally the possible stabilization effect of domain closure on PGK conformation, thermal unfolding experiments with the chemically modified enzyme were devised Pig muscle PGK contains two reactive cysteinyl residues in the hinge region between the two domains, on the outer surface of helix 13 (Fig 4A) These thiol-groups not have any direct role in enzyme activity, yet their chemical modification with a relatively bulky reagent (such as carboxamidomethylation) leads to loss of enzyme activity [85] Small angle X-ray scattering data with this inactive CM-PGK provided evidence for steric prevention of domain closure [34] DSC runs of CM-PGK show significantly wider peaks compared to the unmodified enzyme (Fig 1A 1879 Substrate-assisted domain–domain cooperativity and B), suggesting a less cooperative heat transition In addition, there is a remarkable loss of stability of the whole molecule, as indicated by the large decrease both in the Tm values and in the calorimetric heat (Qt) (Table 1) Plotting the data in the way shown in Fig 2, can be approached by a straight line (not shown) and the activation parameters for thermal unfolding of this modified PGK are clearly different from those for the intact active enzyme (Table 2) The experiment also showed that the intact state of helix 13 is important for the stability of the whole molecule Previous studies with pig muscle PGK suggest that introduction of the chemical label perturbs the conformational state of helix 13, and thereby may also disrupt important stabilizing contacts between the two domains [80] The most interesting part of the results is the protective effects of the substrates against thermal unfolding of CM-PGK Protection by each substrate correlates well with the full substrate binding ability of this modified enzyme [13] The order of effects by each substrate in the binary complexes is the same as found for the unmodified active PGK (Fig 1A and B; Tables and 2) There is, however, a remarkable difference between the modified and unmodified enzymes: the ternary complex of CM-PGK does not exhibit the highest stability among the enzyme–substrate complexes This can only be due to lack of domain closure, as substrate binding to CM-PGK has failed to induce domain closure as shown in a previous small angle X-ray scattering study [34] Thus, our results revealed that the highest conformational stability is a characteristic of the native, functionally competent ternary complex The larger protection by substrates in the ternary complex (vs the binary ones) is abolished in the modified inactive CM-PGK, where domain closure is prevented The increased conformational stability of the closed conformation has been also judged by surveying the crystal structure Taken together, the calorimetric experiments, in accordance with the crystal structures, suggest that the domain-closed conformation is an exclusive characteristic of the ternary complex, i.e both substrates are required for inducing domain closure Experimental procedures Enzymes and chemicals Pig muscle PGK was isolated as described earlier [8] and stored as a microcrystalline suspension in the presence of ammonium sulphate and mm dithiothreitol Yeast PGK was obtained from Sigma (St Louis, MO, USA) as a preci- 1880 A Varga et al pitate in the presence of m ammonium sulphate Their activity was determined using 3-PG and MgATP as substrates in a coupled assay using GAPDH (prepared from pig muscle [86]) as auxiliary enzyme and varied between 300 and 500 katỈmol)1 Two mutants of yeast PGK were constructed: the W308F, W333F, Y122W triple mutant (denoted as W122), and the W308F point mutant (W333) The mutants contain a single Trp residue either in the N- (W122), or the C-terminal domain (W333) The expression and purification of the mutants were described recently [62] Both mutants were His-tagged, and purified using Ni+ affinity chromatography The enzyme activity of the W122 and W333 mutants was measured after purification Neither the His-tag, nor the mutations influenced significantly the catalytic activity The sodium salts of 3-PG, ATP, ADP and NADH were Boehringer products The complexes of MgATP, MgADP were formed by adding MgCl2 (Sigma) in molar excess (12 mm), which assured a practically complete saturation on the basis of the dissociation constants of 0.1 and 0.6 mm, respectively, which were averaged from the literature [87–91] Iodoacetamide was from Sigma All other chemicals were reagent-grade commercial products Preparation of enzyme solutions Crystals of pig muscle PGK or the precipitates of the wildtype yeast PGK were dissolved in 50 mm Tris ⁄ HCl pH 7.5 containing mm EDTA and mm dithiothreitol and dialysed against the same buffer to remove (NH4)2SO4 GAPDH was similarly desalted Dialysis of the yeast PGK mutants was not required, as they were stored in desalted frozen solutions [62] Protein concentration was determined from the UV absorption at 280 nm using the method described by Pace and coworkers [92] The values of 27 900, 21 400, 14 400 and 15 900 m)1Ỉcm)1 were obtained for the pig PGK, the wild-type yeast PGK, W122 and W333 mutants of yeast PGK, respectively The molecular mass of PGK was taken uniformly to be 44.5 kDa Alkylation of pig muscle PGK was performed with iodoacetamide as described previously [85] The reaction was stopped by the addition of 10 mm dithiothreitol and the excess of the reagents was removed by dialysis DSC DSC measurements were performed on a MicroCal VPDSC type microcalorimeter (MicroCal Incorporate, Northampton, MA, USA) with a cell volume of 0.51 mL The applied scanning rates are given in the Figure legends The protein concentration was 0.003 mm (0.13 mgỈmL)1) in all experiments All samples were carefully degassed before the experiments The results were analysed using microcal origin 5.0 software The melting temperature (Tm) was FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS A Varga et al Substrate-assisted domain–domain cooperativity determined after subtraction of the instrumental baseline In the heat induced unfolding experiments carried out in the presence of substrates, high concentrations of ligands (much above the normal saturation) were used It was checked that further increase of ligand concentrations did not affect the results Fluorimetric experiments Fluorescence detected heat induced unfolding was carried out using a SPEX Fluoromax-3 spectrofluorometer equipped with a Peltier thermostat (Edison, NJ, USA) The same heating rate and protein concentration was used as for the DSC Fluorescence was excited at 295 nm and monitored at its emission maximum using nm bandwidths on both sides with a cm path length for excitation and mm for emission While the temperature was increased at a rate of 1°Ỉmin)1, the fluorescence intensity was recorded at every 0.33 The measured fluorescence intensities were converted into the apparent fraction of native protein, FN, vs temperature according to the Eqn (1) [93] FN ẳ IU ỵ mU Tị I IU ỵ mU Tị IN ỵ mN Tị ð1Þ where I is the fluorescence intensity measured at temperature T, and IN and IU are the intercepts and mN and mU are the slopes of the pre- and post-transitional base lines of the raw data, respectively Quantitative analysis of the experimental data Thermal unfolding experiment were analysed according to Sanchez-Ruiz et al [77], by assuming the two-state irreversible model: k NÀ D ! ð2Þ where N and D are the native and the denatured forms of the protein and k is the first order rate constant It was shown that the first order rate constant (k) of this process is related to the shape of the calorimetrically determined heat capacity curve in the following way [77]: k¼ vCp Qt À Q ð3Þ where v is the scanning rate, Cp is the experimental heat capacity, Qt is the total heat of the process (identical to the integrated area under the transition peak) and Q is the heat evolved at a given temperature The rate constants obtained in this way were analysed as a function of the temperature according to the Arrhenius relationship: k ẳ AexpE=RTị 4ị where A is the pre-exponential factor, E is the activation energy, R is the gas constant and T is the absolute tempera- FEBS Journal 272 (2005) 1867–1885 ª 2005 FEBS ture The terms A and E can be expressed in termodynamic quantities: A¼ kB T h and E ẳ DGz 5ị and 6ị where kB is the Boltzmann constant, h is Planck constant and DGà is the activation free enthalpy of the unfolding process Although this expression of the pre-exponential factor is rigorously valid only in the gas phase and neglects the solvent effects, we apply this relationship here as an approximation that affects the absolute maxima of the activation barrier, but probably has little effect on the relative results for the different proteins ⁄ mutants To obtain the activation enthalpy (DHà) and the activation entropy (DSà) of the process separately, the Arrhenius equation can be transformed accordingly: ln k kB DSz DH z ẳ ln ỵ T h R RT 7ị The plot of ln(k ⁄ T) vs ⁄ T gives a straight line, the slope and the intercept on the ordinate give DHà and DSà, respectively The calorimetric data were also analysed according to another type of transformation of the data, as described by Sanchez-Ruiz et al [77]:   Qt E E 8ị ẳ ln ln RTm RT Qt À Q where Tm is the transition temperature The plot of ln[ln(Qt ⁄ Qt–Q)] vs ⁄ T gives a straight line The same equation can be used to analyse the fluorimetrically detected heat transition, since the following relationship holds: FN ẳ ẵN0 Qt ẳ ẵN Qt Q ð9Þ where FN is the fraction of the native protein, as defined by Eqn (1), [N]0 and [N] are the molar fractions of the native state, initially and at any given temperature, respectively Thus, from the fluorimetric data the plot of ln(ln([N]0 ⁄ [N]) against ⁄ T was prepared, on the basis of Eqn (8) Computer molecular graphics For visualizing and analysing the molecular details of the 12 PGK structures studied the insight ii (Biosym ⁄ MSI, San Diego, CA, USA) software was used The pdb codes of the protein co-ordinates used in structural comparisons are as follows: 1PHP (B stearothermophilus PGK*MgADP [9]), 1VJC (pig muscle PGK*MgATP [83]), 1HDI (pig muscle PGK*3PG*MgADP [5]), 1KF0 (pig muscle PGK*3PG* MgAMPPCP [80]), 13PK (T brucei PGK*3PG*MgADP [79]), 1VPE (T maritima PGK*3PG*MgAMPPNP [4]), 3PGK (substrate-free yeast PGK [7]) and 1QPG (mutant yeast PGK*3PG*MgAMPPNP [84]) The pdb co-ordinates 1881 Substrate-assisted domain–domain cooperativity of pig muscle PGK*3PG binary [8], pig muscle PGK*3PG*MnAMPPNP [81], yeast PGK*3-PG binary complex [82] and of the substrate-free pig muscle PGK [80] were obtained from the authors Mapping of the molecular contacts responsible for maintaining the three-dimensional structure of PGK was carried out in three steps First, the conserved residues were selected after making alignments of 168 PGK sequences found in ExPASy Molecular Biology Server As a second, the lists of all peptide H bonds, the hydrophobic, electrostatic and H-bonding contacts of the conserved side chains (besides the contacts between two side chains the contacts including a side chain atom with a backbone atom are also considered) were enumerated separately for all the investigated structures The distance limits for H bonds, hydrophobic and ionic interactions were 3.5, 4.5 and ˚ 4.0 A, respectively In one case, from all type of contacts only those ones were selected which invariantly exist in all 12 structures In the other case, only the variable contacts were considered, characteristic of each of the substrate complexes In the third step the selected contacts were visualized and compared for related pairs of structures superposed according to the core b strands of either the N- or the C-terminal domain PGK structures from different organisms were compared using the structure based sequence alignment, prepared by us [5] and completed here with the data of yeast and Bs PGKs Acknowledgements Thanks are due to K Harlos and A May (Laboratory of Molecular Biophysics, University Oxford, UK), N.R Chandra (Molecular Biophysics Unit, Indian Institute of Sciences, Bangalore, Karnataka, India) ´ and to Z Kovari (Institute of Enzymology, BRC, Hung Acad Sci., Budapest, Hungary) for providing the 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