Éva Gráczer

A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK) is the enzyme responsible for the first ATP-generating step of glycolysis and has been implicated extensively in oncogenesis and its development. Solution small angle x-ray scattering (SAXS) data, in combination with crystal structures of the enzyme in complex with substrate and product analogues, reveal a new conformation for the resting state of the enzyme and demonstrate the role of substrate binding in the preparation of the enzyme for domain closure. Comparison of the x-ray scattering curves of the enzyme in different states with crystal structures has allowed the complete reaction cycle to be resolved both structurally and temporally. The enzyme appears to spend most of its time in a fully open conformation with short periods of closure and catalysis, thereby allowing the rapid diffusion of substrates and products in and out of the binding sites. Analysis of the open apoenzyme structure, defined through deformable elastic network refinement against the SAXS data, suggests that interactions in a mostly buried hydrophobic region may favor the open conformation. This patch is exposed on domain closure, making the open conformation more thermodynamically stable. Ionic interactions act to maintain the closed conformation to allow catalysis. The short time PGK spends in the closed conformation and its strong tendency to rest in an open conformation imply a spring-loaded release mechanism to regulate domain movement, catalysis, and efficient product release.

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.

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 β‐strand L, under the concerted action of both substrates.

Insight into the Mechanism of Domain Movements and their Role in Enzyme Function: Example of 3-Phosphoglycerate Kinase

Coupling of structural flexibility and biological function is an essential feature of proteins. The role of relative domain movements in enzyme function has been evidenced in many cases. However, the way of communication between protein domains and its manifestation in their movements as well as in the biological function are rarely delineated. In this review we summarize comprehensive studies with a typical hinge-bending two-domain enzyme, 3-phosphoglycerate kinase. A possible mechanism is proposed by which the two substrates that bind to different domains trigger the operation of the molecular hinges, located in the interdomain region. Various crystal structures of the enzyme have been determined with different relative domain positions, suggesting that domain closure brings the two substrates together for the catalysis. Substrate-caused conformational changes in the binary and the ternary complexes have been tested with the solubilized enzyme using physical methods, such as differential scanning calorimetry, small angle X-ray scattering and infrared spectroscopy. The results indicated the existence of strong cooperativity between the two domains and that the presence of both substrates is necessary for the domain closure. Comparison of the atomic contacts in the structures has led to selection of conserved side-chains, which may be involved in the domain movement. On this basis a hypothesis was put forward about the molecular mechanism of interdomain co-operation. Enzyme kinetic, ligand binding and small angle X-ray scattering studies with various site-directed mutants have confirmed this hypothesis. Namely, a special H-bonding network (a double molecular switch) seems to be responsible for operation of the main molecular hinge at the beta-strand L under the concerted action of both substrates.

Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis

The three‐dimensional structure of the enzyme 3‐isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn2+, its substrate isopropylmalate and its co‐factor product NADH at 2.0 Å resolution features a fully closed conformation of the enzyme. Upon closure of the two domains, the substrate and the co‐factor are brought into precise relative orientation and close proximity, with a distance between the C2 atom of the substrate and the C4N atom of the pyridine ring of the co‐factor of approximately 3.0 Å. The structure further shows binding of a K+ ion close to the active site, and provides an explanation for its known activating effect. Hence, this structure is an excellent mimic for the enzymatically competent complex. Using high‐level QM/MM calculations, it may be demonstrated that, in the observed arrangement of the reactants, transfer of a hydride from the C2 atom of 3‐isopropylmalate to the C4N atom of the pyridine ring of NAD+ is easily possible, with an activation energy of approximately 15 kcal·mol−1. The activation energy increases by approximately 4–6 kcal·mol−1 when the K+ ion is omitted from the calculations. In the most plausible scenario, prior to hydride transfer the ε‐amino group of Lys185 acts as a general base in the reaction, aiding the deprotonation reaction of 3‐isopropylmalate prior to hydride transfer by employing a low‐barrier proton shuttle mechanism involving a water molecule.

Atomic level description of the domain closure in a dimeric enzyme: Thermus thermophilus 3-isopropylmalate dehydrogenase

The domain closure associated with the catalytic cycle is described at an atomic level, based on pairwise comparison of the X-ray structures of homodimeric Thermus thermophilus isopropylmalate dehydrogenase (IPMDH), and on their detailed molecular graphical analysis. The structures of the apo-form without substrate and in complex with the divalent metal-ion to 1.8 Å resolution, in complexes with both Mn(2+) and 3-isopropylmalate (IPM), as well as with both Mn(2+) and NADH, were determined at resolutions ranging from 2.0 to 2.5 Å. Single crystal microspectrophotometric measurements demonstrated the presence of a functionally competent protein conformation in the crystal grown in the presence of Mn(2+) and IPM. Structural comparison of the various complexes clearly revealed the relative movement of the two domains within each subunit and allowed the identification of two hinges at the interdomain region: hinge 1 between αd and βF as well as hinge 2 between αh and βE. A detailed analysis of the atomic contacts of the conserved amino acid side-chains suggests a possible operational mechanism of these molecular hinges upon the action of the substrates. The interactions of the protein with Mn(2+) and IPM are mainly responsible for the domain closure: upon binding into the cleft of the interdomain region, the substrate IPM induces a relative movement of the secondary structural elements βE, βF, βG, αd and αh. A further special feature of the conformational change is the movement of the loop bearing the amino acid Tyr139 that precedes the interacting arm of the subunit. The tyrosyl ring rotates and moves by at least 5 Å upon IPM-binding. Thereby, new hydrophobic interactions are formed above the buried isopropyl-group of IPM. Domain closure is then completed only through subunit interactions: a loop of one subunit that is inserted into the interdomain cavity of the other subunit extends the area with the hydrophobic interactions, providing an example of the cooperativity between interdomain and intersubunit interactions.

Symmetrical refolding of protein domains and subunits: Example of the dimeric two-domain 3-isopropylmalate dehydrogenases

The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. I5 (Vib), is described. In all three cases, instead of a self-template mechanism, the high extent of symmetry and cooperativity in folding of subunits and domains have been concluded from the following experimental findings: The complex time course of refolding, monitored by Trp fluorescence, consists of a fast (the rate constant varies as 16.5, 25.0, and 11.7 min-1 in the order of Tt, Ec, and Vib IPMDHs) and a slow (the rate constants are 0.11, 0.80, and 0.23 min-1 for the three different species) first-order process. However, a burst increase of Trp fluorescence anisotropy to the value of the native states indicates that in all three cases the association of the two polypeptide chains occurs at the beginning of refolding. This dimeric species binds the substrate IPM, but the native-like interactions of the tertiary and quaternary structures are only formed during the slow phase of refolding, accompanied by further increase of protein fluorescence and appearance of FRET between Trp side chain(s) and the bound NADH. Joining the contacting arms of each subunit also takes place exclusively during this slow phase. To monitor refolding of each domain within the intact molecule of T. thermophilus IPMDH, Trps (located in separate domains) were systematically replaced with Phes. The refolding processes of the mutants were followed by measuring changes in Trp fluorescence and in FRET between the particular Trp and NADH. The high similarity of time courses (both in biphasicity and in their rates) strongly suggests cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.

Nucleotide promiscuity of 3-phosphoglycerate kinase is in focus: implications for the design of better anti-HIV analogues

The wide specificity of 3-phosphoglycerate kinase (PGK) towards its nucleotide substrate is a property that allows contribution of this enzyme to the effective phosphorylation (i.e. activation) of nucleotide-based pro-drugs against HIV. Here, the structural basis of the nucleotide-PGK interaction is characterised in comparison to other kinases, namely pyruvate kinase (PK) and creatine kinase (CK), by enzyme kinetic analysis and structural modelling (docking) studies. The results provided evidence for favouring the purine vs. pyrimidine base containing nucleotides for PGK rather than for PK or CK. This is due to the exceptional ability of PGK in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate-chain for catalysis. As for the D-/L-configurations of the nucleotides, the L-forms (both purine and pyrimidine) are well accepted by PGK rather than either by PK or CK. Here again the dominance of the hydrophobic interactions of the L-form of pyrimidines with PGK is underlined in comparison with those of PK or CK. Furthermore, for the l-forms, the absence of the ribose OH-groups with PGK is better tolerated for the purine than for the pyrimidine containing compounds. On the other hand, the positioning of the phosphate-chain is an even more important term for PGK in the case of both purines and pyrimidines with an L-configuration, as deduced from the present kinetic studies with various nucleotide-site mutants of PGK. These characteristics of the kinase-nucleotide interactions can provide a guideline for designing new drugs.

Transient kinetic studies reveal isomerization steps along the kinetic pathway of Thermus thermophilus 3-isopropylmalate dehydrogenase

To identify the rate‐limiting step(s) of the 3‐isopropylmalate dehydrogenase‐catalysed reaction, time courses of NADH production were followed by stopped flow (SF) and quenched flow (QF). The steady state kcat and Km values did not vary between enzyme concentrations of 0.1 and 20 μm. A burst phase of NADH formation was shown by QF, indicating that the rate‐limiting step occurs after the redox step. The kinetics of protein conformational change(s) induced by the complex of 3‐isopropylmalate with Mg2+ were followed by using the fluorescence resonance energy transfer signal between protein tryptophan(s) and the bound NADH. A reaction scheme was proposed by incorporating the rate constant of a fast protein conformational change (possibly domain closure) derived from the separately recorded time‐dependent formation of the fluorescence resonance energy transfer signal. The rate‐limiting step seems to be another slower conformational change (domain opening) that allows product release.

Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase.

X‐ray structures of 3‐isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal‐ion in the metal–IPM assisted domain closure. Here solution studies were carried out to test its importance. Small‐angle X‐ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0–5%) extent of domain closure in the absence of the metal‐ion. Only the metal–IPM complex, but neither the metal‐ion nor the free IPM itself, is efficient in stabilizing the native protein conformation as confirmed by denaturation experiments with Escherichia coli IPMDH and by studies of the characteristic fluorescence resonance energy transfer (FRET) signal (from Trp to bound NADH) with both IPMDHs. A possible atomic level explanation of the metal‐effect is given.