Thermodynamic analysis of substrate induced domain closure of 3-phosphoglycerate kinase

The energetic changes accompanying domain closure of 3-phosphoglycerate kinase, a typical hinge-bending enzyme, were assessed. Calorimetric titrations of the enzyme with each substrate, both in the absence and presence of the other one, provide information not only about the energetics of substrate binding, but of the associated conformational changes, including domain closure. Our results suggest that conformational rearrangements in the hinge generated by binding of both substrates provide the main driving force for domain closure overcoming the slightly unfavourable contact interactions between the domains.     

Substrate-induced double sided H-bond network as a means of domain closure in 3-phosphoglycerate kinase

Closure of the two domains of 3-phosphoglycerate kinase, upon substrate binding, is essential for the enzyme function. The available crystal structures cannot provide sufficient information about the mechanism of substrate assisted domain closure and about the requirement of only one or both substrates, since lattice forces may hinder the large scale domain movements. In this study the known X-ray data, obtained for the open and closed conformations, were probed by solution small-angle X-ray scattering experiments. The results prove that binding of both substrates is essential for domain closure. Molecular graphical analysis, indeed, reveals formation of a double-sided H-bond network, which affects substantially the shape of the main molecular hinge at beta-strand L, under the concerted action of both substrates.     

Identification of specific interactions that drive ligand-induced closure in five enzymes with classic domain movements

In order to better understand ligand-induced closure in domain enzymes, open unliganded X-ray structures and closed liganded X-ray structures have been studied in five enzymes: adenylate kinase, aspartate aminotransferase, citrate synthase, liver alcohol dehydrogenase, and the catalytic subunit of cAMP-dependent protein kinase. A sequential model of ligand binding and domain closure was used to test the hypothesis that the ligand actively drives closure from an open conformation. The analysis supports the assumption that each enzyme has a dedicated binding domain to which the ligand binds first and a closing domain. In every case, a small number of residues are identified to interact with the ligand to initiate and drive domain closure. In all cases except adenylate kinase, the backbone of residues located in an interdomain-bending region (hinge site) is identified to interact with the ligand to aid in driving closure. In adenylate kinase, the side-chain of a residue located directly adjacent to a bending region drives closure. It is thought that by binding near a hinge site the ligand is able to get within interaction range of residues when the enzyme is in the open conformation. Interdomain bending regions not involved in inducing closure are involved in control, helping to determine the location of the hinge axis. Similarities have been discovered between aspartate aminotransferase and citrate synthase that only come to light in the context of their dynamical behaviour in response to binding their substrate. Similarity also exists between liver alcohol dehydrogenase and cAMP-dependent protein kinase whereby groups on NAD and ATP, respectively, mimic the backbone of a single amino acid residue in a process where a three residue segment located at the terminus of a beta-sheet, moves to form hydrogen bonds with the mimic that resemble those found in a parallel beta-sheet. This interaction helps to drive domain closure in a process that has analogy to protein folding.     

Role of side-chains in the operation of the main molecular hinge of 3-phosphoglycerate kinase

The single mutants (F165A, E192A, F196A, S392A, T393A) at and near the main hinge (beta-strand L) of human 3-phosphoglycerate kinase (hPGK) exhibit variously reduced enzyme activity, indicating the cumulative effects of these residues in regulating domain movements. The residues F165 and E192 are also essential in maintaining the conformational integrity of the whole molecule, including the hinge-region. Shortening of betaL by deleting T393 has led to a dramatic activity loss and the concomitant absence of domain closure (as detected by small angle X-ray scattering), demonstrating the role of betaL in functioning of hPGK. The role of each residue in the conformational transmission is described.     

Correlation between conformational stability of the ternary enzyme-substrate complex and domain closure of 3-phosphoglycerate kinase

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 Biochem180, 61-66]. Thus, conformational stability correlates with domain closure that requires simultaneous binding of both substrates.     

Phosphorylation- and Nucleotide-Binding-Induced Changes to the Stability and Hydrogen Exchange Patterns of JNK1β1 Provide Insight into Its Mechanisms of Activation

Many studies have characterized how changes to the stability and internal motions of a protein during activation can contribute to their catalytic function, even when structural changes cannot be observed. Here, unfolding studies and hydrogen–deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1β1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide binding on both inactive and active JNK1β1 using the ATP analogue, 5?-adenylyl-imidodiphosphate (AMP-PNP). JNK1β1 phosphorylation alters HX in regions involved in catalysis and substrate binding, changes that can be ascribed to functional modifications in either structure and/or backbone flexibility. Increased HX in the hinge between the N- and C-terminal domains implied that it acquires enhanced flexibility upon phosphorylation that may be a prerequisite for interdomain closure. In combination with the finding that nucleotide binding destabilizes the kinase, the patterns of solvent protection by AMP-PNP were consistent with a novel mode of nucleotide binding to the C-terminal domain of a destabilized and open domain conformation of inactive JNK1β1. Solvent protection by AMP-PNP of both N- and C-terminal domains in active JNK1β1 revealed that the domains close around nucleotide upon phosphorylation, concomitantly stabilizing the kinase. This suggests that phosphorylation activates JNK1β1 in part by increasing hinge flexibility to facilitate interdomain closure and the creation of a functional active site. By uncovering the complex interplay that occurs between nucleotide binding and phosphorylation, we present new insight into the unique mechanisms by which JNK1β1 is regulated.     

Nucleotide binding to pig muscle 3-phosphoglycerate kinase in the crystal and in solution: relationship between substrate antagonism and interdomain communication.

Binding constants for the nucleotide substrates were determined in two different crystalline forms of pig muscle 3-phosphoglycerate kinase (PGK): the binary complex with 3-phosphoglycerate (3-PG) in which the two domains are in an open conformation (Harlos, Vas, and Blake (1992) Proteins, 12, 133-144) and the ternary complex with 3-PG and the Mg salt of the ATP analogue, beta,gamma-methyleneadenosine-5'-triphosphate (AMP-PCP), the structure of which is under resolution. Competitive titrations have been performed in the presence of the chromophoric analogue of ATP, 2'3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP), similar to those previously carried out in solution, where a weakening of the binding of the nucleotide substrates in the presence of the other substrate, 3-PG, has been observed (Vas, Merli, and Rossi (1994) Biochem. J. 301, 885-891). Here the K(d) values for MgADP were found to be 0.096 +/- 0.021 and 0.045 +/- 0.016 mM, respectively, for the crystals of the binary and ternary complexes. Both K(d) values are significantly smaller than the one obtained in solution in the presence of 3-PG (0.38 +/- 0.05 mM) and are close to the values determined in solution in the absence of 3-PG (0.06 +/- 0.01 mM). Thus, the "substrate antagonism" observed in solution is not present in either of the investigated crystal forms. Further nucleotide binding studies with the solubilized enzyme have shown that 3-PG has no effect on ADP (Mg(2+)-free) binding (K(d) = 0.34 +/- 0.05 mM), while it weakens MgADP binding. Thus, 3-PG abolishes the strengthening effect of the Mg(2+) ion on the binding of ADP. This phenomenon is apparently due to the interaction between the carboxyl group of 3-PG and the protein, since the carboxyl-lacking analogue glycerol-3-phosphate has no detectable effect on MgADP binding. Comparison of the crystallographic data of different PGK binary (with either 3-PG or MgADP) and ternary (with both 3-PG and MgADP) complexes, having open and closed conformations, respectively, provides a possible structural explanation of the substrate antagonism. We suggest that the specific interaction between the 3-PG carboxylic group and a conserved arginine side chain is changed during domain closure, and, through interdomain communication, this change may be transmitted to the site in which Mg(2+) binds the ADP phosphates. This effect is abolished in the crystals of pig muscle PGK, in which lattice forces stabilize the open domain conformation.     

NMR analysis of site-specific mutants of yeast phosphoglycerate kinase. An investigation of the triose-binding site

Site-specific mutants of yeast phosphoglycerate kinase have been produced in order to investigate the roles of the 'basic-patch' residues, arginine 168 and histidine 170. The fully-conserved residue, arginine 168, has been replaced with a lysine (R168K) and a methionine (R168M) residue, while the non-conserved histidine 170 has been replaced with an aspartate (H170D). Comparison of the 500-MHz 1H-NMR spectra of the mutant proteins with that of wild-type phosphoglycerate kinase shows that the overall fold of the mutants remains essentially unaltered from that of the native enzyme. Results of NOE experiments indicate that there are only very minor changes in structure in the vicinity of the mutations. These mutations have also led to firm sequence-specific resonance assignments to histidines 62, 167 and 170. NMR studies of 3-phosphoglycerate binding show that decreasing the positive charge in the sequence 168-170 reduces the binding of this substrate (by about 15-fold and 4-fold for mutants R168M and H170D respectively). Mutant R168K binds 3-phosphoglycerate with an affinity about twofold less than that of the native enzyme. Significantly, the activity of mutant H170D, measured at saturating substrate concentrations, is unchanged from that of the wild-type enzyme. This indicates that this residue is not of major importance in the binding or reaction of 3-phosphoglycerate. The observation is in agreement with results obtained for the wild-type enzyme, which indicate that 3-phosphoglycerate interacts most strongly with histidine 62 and least strongly with histidine 170, as would be predicted from the X-ray crystal structure. Substitution of positively charged arginine 168 with neutral methionine (or positively charged lysine) does not cause a detectable change in the pKa values of the neighbouring histidine groups, in as much as they remain below 3. The results reported here indicate that the observed reduction in catalytic efficiency relates less to direct electrostatic effects than to the mutants' inability to undergo 3-phosphoglycerate-induced conformational changes.     

Stability domains, substrate-induced conformational changes, and hinge-bending motions in a psychrophilic phosphoglycerate kinase. A microcalorimetric study

The cold-active phosphoglycerate kinase from the Antarctic bacterium Pseudomonas sp. TACII18 exhibits two distinct stability domains in the free, open conformation. It is shown that these stability domains do not match the structural N- and C-domains as the heat-stable domain corresponds to about 80 residues of the C-domain, including the nucleotide binding site, whereas the remaining of the protein contributes to the main heat-labile domain. This was demonstrated by spectroscopic and microcalorimetric analyses of the native enzyme, of its mutants, and of the isolated recombinant structural domains. It is proposed that the heat-stable domain provides a compact structure improving the binding affinity of the nucleotide, therefore increasing the catalytic efficiency at low temperatures. Upon substrate binding, the enzyme adopts a uniformly more stable closed conformation. Substrate-induced stability changes suggest that the free energy of ligand binding is converted into an increased conformational stability used to drive the hinge-bending motions and domain closure.     

Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase

Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP gamma-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val(93), Leu(122), Phe(179), and Pro(232) in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with K(m) values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe(179) is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.     

Correlation between enzyme activity and hinge-bending domain displacement in 3-phosphoglycerate kinase

Diffuse X-ray-scattering data give evidence for large-scale structural change in pig muscle 3-phosphoglycerate kinase upon substrate binding. Simultaneous binding of 3-phosphoglycerate and MgATP either to the unmodified enzyme or to its active methylated derivative leads to about an 0.1-nm decrease in radius of gyration. These data coincide well with the previous data for yeast 3-phosphoglycerate kinase. When, instead of methylation, the two reactive thiol groups of pig muscle 3-phosphoglycerate kinase are carboxamidomethylated, the enzyme becomes inactive and the radii of gyration of its 'apo' and 'holo' forms do not differ within limits of experimental error. Thus, a correlation exists between the activity of 3-phosphoglycerate kinase and its substrate-induced large-scale conformational change. This correlation is a strong argument in favor of the functional importance of domain locking in the reaction catalyzed by 3-phosphoglycerate kinase.     

Structural analysis of substrate and effector binding in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase

The crystal structure of Mycobacterium tuberculosis d-3-phosphoglycerate dehydrogenase has been solved with bound effector, l-serine, and substrate, hydroxypyruvic acid phosphate, at resolutions of 2.7 and 2.4 A, respectively. The subunits display the same extreme asymmetry as seen in the apo-structure and provide insight into the mode of serine binding and closure of the active site. Mutagenesis studies confirm the identity of the main residues involved in serine binding and suggest that the poly glycine stretch in the loop that contains the locus for the 160 degrees rotation that leads to subunit asymmetry may have a larger role in folding than in catalysis. The lack of electron density for the cofactor, NADH, in any of the crystals examined led us to study binding by stopped flow kinetic analysis. The kinetic data suggest that productive NADH binding, that would support catalytic turnover, is dependent on the presence of substrate. This observation, along with the binding of substrate in the active site, but in an unproductive conformation, suggests a possible mechanism where initial binding of substrate leads to enhanced interaction with cofactor accompanied by a rearrangement of catalytically critical residue side chains. Furthermore, comparison to the structure of a truncated form of human d-3-phosphoglycerate dehydrogenase with cofactor and a substrate analog, provides insight into the conformational changes that occur during catalysis.     

Structural Tightening and Interdomain Communication in the Catalytic Cycle of Phosphoglycerate Kinase

Changes in amide-NH chemical shift and hydrogen exchange rates as phosphoglycerate kinase progresses through its catalytic cycle have been measured to assess whether they correlate with changes in hydrogen bonding within the protein. Four representative states were compared: the free enzyme, a product complex containing 3-phosphoglyceric acid (3PG), a substrate complex containing ADP and a transition-state analogue (TSA) complex containing a 3PG-AlF₄⁻-ADP moiety. There are an overall increases in amide protection from hydrogen exchange when the protein binds the substrate and product ligands and an additional increase when the TSA complex is formed. This is consistent with stabilisation of the protein structure by ligand binding. However, there is no correlation between the chemical shift changes and the protection factor changes, indicating that the protection factor changes are not associated with an overall shortening of hydrogen bonds in the protected ground state, but rather can be ascribed to the properties of the high-energy, exchange-competent state. Therefore, an overall structural tightening mechanism is not supported by the data. Instead, we observed that some cooperativity is exhibited in the N-domain, such that within this domain the changes induced upon forming the TSA complex are an intensification of those induced by binding 3PG. Furthermore, chemical shift changes induced by 3PG binding extend through the interdomain region to the C-domain β-sheet, highlighting a network of hydrogen bonds between the domains that suggests interdomain communication. Interdomain communication is also indicated by amide protection in one domain being significantly altered by binding of substrate to the other, even where no associated change in the structure of the substrate-free domain is indicated by chemical shifts. Hence, the communication between domains is also manifested in the accessibility of higher-energy, exchange-competent states. Overall, the data that are consistent with structural tightening relate to defined regions and are close to the 3PG binding site and in the hinge regions of 3-phosphoglycerate kinase.     

Structure-function relationships in 3-phosphoglycerate kinase: role of the carboxy-terminal peptide

Yeast 3-phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr approximately 45,000) composed of two globular domains. Each domain corresponds approximately to the amino- and carboxy-terminal halves of the polypeptide chain. The carboxy-terminal end extends over the interdomain "hinge" region and packs against the amino-terminal domain. It has been proposed that domain movement, resulting in closure of the active site cleft, is essential for the catalytic function of PGK. Large-scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site-specific mutagenesis, we have removed a 15-amino-acid carboxy-terminal fragment, in order to probe its role in the substrate- and sulfate-induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfate-induced activation. The Km for ATP was essentially unchanged (Km = 0.23 mM) in comparison to the native enzyme (Km = 0.30 mM), whereas the Km value for 3-phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy-terminal segment is important for the mechanism of the substrate- and sulfate-induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy-terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy-terminal peptide also contributes to the stability of PGK.     

Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP.

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.     

A proton-NMR study of a site-directed mutation (His388----Glu) in the interdomain region of yeast phosphoglycerate kinase. Implications for domain movement

Proton NMR has been used to study a site-directed mutant of yeast phosphoglycerate kinase in which the interdomain residue His388 has been replaced by a glutamine residue. Using 1H-NMR spectroscopy, it was found that 3-phosphoglycerate binding to the mutant protein induces different conformational effects to those observed for the wild-type enzyme. These differences are not only located at the 3-phosphoglycerate binding site but are also seen as long-range effects at the surface of the protein. Measurements of the Kd for 3-phosphoglycerate from the NMR experiments show that the mutant enzyme has a 30-times reduced affinity for this substrate as compared with the wild-type enzyme. These data are consistent with the suggestion that an aromatic residue at position 388 plays an important role in the proposed hinge-bending mechanism.     

Transient kinetic analysis of L-serine interaction with Escherichia coli D-3-phosphoglycerate dehydrogenase containing amino acid mutations in the hinge regions

In Escherichia colid-3-phosphoglycerate dehydrogenase, the amino acid sequences G294-G295 and G336-G337 are found between structural domains and appear to function as hinge regions. Mutagenesis studies of these sequences showed that bulky side chains had significant effects on the kinetic properties of the enzyme. Placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady-state analysis has demonstrated that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-336 to valine has its main effect on the Kd of serine binding to one form of the free enzyme (E) while maintaining the cooperativity of binding observed in the native enzyme. Conversion of Gly-294 to valine eliminates a rate limiting conformational change that follows serine binding to E. The conformational change between the two forms of free enzyme is maintained, but the Hill coefficient for cooperativity is significantly lowered. The data indicate that the cooperative transmission induced by serine binding is transmitted through the Gly294-Gly295 hinge region to the opposite serine binding interface and that this is most likely propagated by way of the substrate binding domain-regulatory domain interface. In the G294 mutant enzyme, both serine bound species, E·Ser and E*·Ser, are present in significant amounts indicating that cooperativity of serine binding does not result from the binding to two different forms. The data also suggest that the E* form may be inactive even when serine is not bound.     

Extracellular-Regulated Kinase 2 Is Activated by the Enhancement of Hinge Flexibility

Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.     

Yeast 3-phosphoglycerate kinase. Essential arginyl residues at the 3-phosphoglycerate binding site.

Yeast 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phospho-transferase, EC 2.7.2.3) is inactivated by phenylglyoxal. Loss of activity correlates with the modification of two arginyl residues, both of which are protected by all of the substrates. The modification is not accompanied by any significant conformational change as determined by optical rotatory dispersion. Ultraviolet difference spectrophotometry indicates that the inactivated enzyme retains its capacity for binding the nucleotide substrates whereas the spectral perturbation characteristic of 3-phosphoglycerate binding is abolished in the modified enzyme. The data suggest that at least one of the two essential arginyl residues is located at or near the 3-phosphoglycerate binding site. A likely role of this residue could be its interaction with the negatively charged phosphate or carboxylate groups of 3-phosphoglycerate.