Ligand-induced conformational rearrangements regulate the switch between membrane-proximal and distal functions of Rho kinase 2.

Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Its functions can be divided into two categories: membrane-proximal and membrane-distal. A recent study concluded that membrane-distal functions require the fully extended conformation, and this conclusion was supported by electron microscopy. The present solution small-angle X-ray scattering (SAXS) study revealed that ROCK2 population is a dynamic mixture of folded and partially extended conformers. Binding of RhoA to the coiled-coil domain shifts the equilibrium towards the partially extended state. Enzyme activity measurements suggest that the binding of natural protein substrates to the kinase domain breaks up the interaction between the N-terminal kinase and C-terminal regulatory domains, but smaller substrate analogues do not. The present study reveals the dynamic behaviour of this long, dimeric molecule in solution, and our structural model provides a mechanistic explanation for a set of membrane-proximal functions while allowing for the existence of an extended conformation in the case of membrane-distal functions.

A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts.

We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.

Dual Role of the Active Site Residues of Thermus thermophilus 3-Isopropylmalate Dehydrogenase: Chemical Catalysis and Domain Closure.

The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the kcat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (Km values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1-13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme-substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure.

Glutamate 270 plays an essential role in K(+)-activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase.

The mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced (∼1%) catalytic activity and negligible activation by K(+) compared to the wild-type enzyme. A 3-4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K(+). SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K(+) into close proximity of the nicotinamide ring of NAD(+) and the electron-withdrawing effect of K(+) may help to polarise the aromatic ring in order to aid the hydride-transfer.

Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis.

UNLABELLED: The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn(2+) , 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. DATABASE: Structural data have been submitted to the Protein Data Bank under accession number 4F7I.

Drugs against Mycobacterium tuberculosis 3-isopropylmalate dehydrogenase can be developed using homologous enzymes as surrogate targets.

3-Isopropylmalate dehydrogenase (IPMDH) from Mycobacterium tuberculosis (Mtb) may be a target for specific drugs against this pathogenic bacterium. We have expressed and purified Mtb IPMDH and determined its physicalchemical and enzymological properties. Size-exclusion chromatography and dynamic light scattering measurements (DLS) suggest a tetrameric structure for Mtb IPMDH, in contrast to the dimeric structure of most IPMDHs. The kinetic properties (kcat and Km values) of Mtb IPMDH and the pH-dependence of kcat are very similar to both Escherichia coli (Ec) and Thermus thermophilus (Tt) IPMDHs. The stability of Mtb IPMDH in 8 M urea is close to that of the mesophilic counterpart, Ec IPMDH, both of them being much less stable than the thermophilic (Tt) enzyme. Two known IPMDH inhibitors, O-methyl oxalohydroxamate and 3-methylmercaptomalate, have been synthesised. Their inhibitory effects were found to be independent of the origin of IPMDHs. Thus, experiments with either Ec or Tt IPMDH would be equally relevant for designing specific inhibitory drugs against Mtb IPMDH.

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 Mg(2+) 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.

Selectivity of kinases on the activation of tenofovir, an anti-HIV agent.

Nucleoside analogues, used in HIV-therapy, need to be phosphorylated by cellular enzymes in order to become potential substrates for HIV reverse transcriptase. After incorporation into the viral DNA chain, because of lacking of their 3'-hydroxyl groups, they stop the elongation process and lead to the death of the virus. Phosphorylation of the HIV-drug derivative, tenofovir monophosphate was tested with the recombinant mammalian nucleoside diphosphate kinase (NDPK), 3-phosphoglycerate kinase (PGK), creatine kinase (CK) and pyruvate kinase (PK). Among them, only CK was found to phosphorylate tenofovir monophosphate with a reasonable rate (about 45-fold lower than with its natural substrate, ADP), while PK exhibits even lower, but still detectable activity (about 1000-fold lower compared to the value with ADP). On the other hand, neither NDPK nor PGK has any detectable activity on tenofovir monophosphate. The absence of activity with PGK is surprising, since the drug tenofovir competitively inhibits both CK and PGK towards their nucleotide substrates, with similar inhibitory constants, K(I) of 2.9 and 4.8mM, respectively. Computer modelling (docking) of tenofovir mono- or diphosphate forms to these four kinases suggests that the requirement of large-scale domain closure for functioning (as for PGK) may largely restrict their applicability for phosphorylation/activation of pro-drugs having a structure similar to tenofovir monophosphate.

Importance of aspartate residues in balancing the flexibility and fine-tuning the catalysis of human 3-phosphoglycerate kinase.

The exact role of the metal ion, usually Mg(2+), in the catalysis of human 3-phosphoglycerate kinase, a well-studied two-domain enzyme, has not been clarified. Here we have prepared single and double alanine mutants of the potential metal-binding residues, D374 and D218. While all mutations weaken the catalytic interactions with Mg(2+), they surprisingly strengthen binding of both MgADP and MgATP, and the effects are even more pronounced for ADP and ATP. Thermodynamic parameters of binding indicate an increase in the binding entropy as a reason for the strengthening. In agreement with the experimental results, computer-simulated annealing calculations for the complexes of these mutants have supported the mobility of the nucleotide phosphates and, as a consequence, formation of their new interaction(s) within the active site. A similar type of mobility is suggested to be a characteristic feature of the nucleotide site of the wild-type enzyme, too, both in its inactive open conformation and in the active closed conformation. This mobility of the nucleotide phosphates that is regulated by the aspartate side chains of D218 and D374 through the complexing Mg(2+) is suggested to be essential in enzyme function.

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.

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.

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 ßF as well as hinge 2 between αh and ßE. 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 ßE, ßF, ßG, 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.

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.

Crystallization and preliminary X-ray diffraction analysis of various enzyme-substrate complexes of isopropylmalate dehydrogenase from Thermus thermophilus.

The Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) enzyme catalyses the penultimate step of the leucine-biosynthesis pathway. It converts (2R,3S)-3-isopropylmalate to (2S)-2-isopropyl-3-oxosuccinate in the presence of divalent Mg(2+) or Mn(2+) and with the help of NAD(+). In order to elucidate the detailed structural and functional mode of the enzymatic reaction, crystals of Tt-IPMDH were grown in the presence of various combinations of substrate and/or cofactors. Here, the crystallization, data collection and preliminary crystallographic analyses of six such complexes are reported.

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, Trp's (located in separate domains) were systematically replaced with Phe's. 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.

Rates of unfolding, rather than refolding, determine thermal stabilities of thermophilic, mesophilic, and psychrotrophic 3-isopropylmalate dehydrogenases.

The relationship between the thermal stability of proteins and rates of unfolding and refolding is still an open issue. The data are very scarce, especially for proteins with complex structure. Here, time-dependent denaturation-renaturation experiments on Thermus thermophilus, Escherichia coli, and Vibrio sp. I5 3-isopropylmalate dehydrogenases (IPMDHs) of different heat stabilities are presented. Unfolding, as monitored by several methods, occurs in a single first-order step with half-times of approximately 1 h, several minutes, and few seconds for the thermophilic, mesophilic, and psychrotrophic enzymes, respectively. The binding of Mn*IPM (the manganese complex of 3-isopropylmalate) markedly reduces the rates of unfolding; this effect is more prominent for the less stable enzyme variants. Refolding is a two-step or multistep first-order process involving an inactive intermediate(s). The restoration of the native structure and reactivation take place with a half-time of a few minutes for all three IPMDHs. Thus, the comparative experimental unfolding-refolding studies of the three IPMDHs with different thermostabilities have revealed a close relationship between thermostability and unfolding rate. Structural analysis has shown that the differences in the molecular contacts between selected nonconserved residues are responsible for the different rates of unfolding. On the other hand, the folding rates might be correlated with the absolute contact order, which does not significantly vary between IPMDHs with different thermostabilities. On the basis of our observations, folding rates appear to be dictated by global structural characteristics (such as native topology, i.e., contact order) rather than by thermodynamic stability.

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.

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.