The structure of Plasmodium falciparum hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase reveals the basis of sulfa resistance.

The clinical efficacy of sulfa drugs as antimalarials has declined owing to the evolution of resistance in Plasmodium falciparum (Pf) malaria parasites. In order to understand the basis of this resistance and to design more effective antimalarials, we have solved 13 structures of the bifunctional enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK)-dihydropteroate synthase (DHPS) from wild-type (WT) P. falciparum and sulfa-resistant mutants, both as apoenzyme and as complexes with pteroate (PTA) and sulfa derivatives. The structures of these complexes show that PTA, which effectively inhibits both the WT and mutants, stays in active sites without steric constraint. In contrast, parts of the sulfa compounds situated outside of the substrate envelope are in the vicinity of the resistance mutations. Steric conflict between compound and mutant residue along with increased flexibility of loop D2 in the mutants can account for the reduced compound binding affinity to the mutants. Kinetic data show that the mutants have enhanced enzyme activity compared with the WT. These PfDHPS structural insights are critical for the design of novel, substrate envelope-compliant DHPS inhibitors that are less vulnerable to resistance mutations. DATABASES: The data reported in this paper have been deposited in the Protein Data Bank, www.wwpdb.org. PDB ID codes: 6JWQ for apoWT; 6JWR, 6JWS, and 6JWT for PTA complexes of WT, A437G (3D7), and V1/S; 6JWU, 6JWV, and 6JWW for STZ-DHP complexes of WT, 3D7, and V1/S; 6JWX, 6JWY, and 6JWZ for SDX-DHP complexes of WT, 3D7, and W2; 6KCK, 6KCL, and 6KCM for Pterin/pHBA complexes of WT, TN1, and W2.

Thiamine deficiency contributes to synapse and neural circuit defects.

BACKGROUND: The previous studies have demonstrated the reduction of thiamine diphosphate is specific to Alzheimer's disease (AD) and causal factor of brain glucose hypometabolism, which is considered as a neurodegenerative index of AD and closely correlates with the degree of cognitive impairment. The reduction of thiamine diphosphate may contribute to the dysfunction of synapses and neural circuits, finally leading to cognitive decline. RESULTS: To demonstrate this hypothesis, we established abnormalities in the glucose metabolism utilizing thiamine deficiency in vitro and in vivo, and we found dramatically reduced dendrite spine density. We further detected lowered excitatory neurotransmission and impaired hippocampal long-term potentiation, which are induced by TPK RNAi in vitro. Importantly, via treatment with benfotiamine, Aß induced spines density decrease was considerably ameliorated. CONCLUSIONS: These results revealed that thiamine deficiency contributed to synaptic dysfunction which strongly related to AD pathogenesis. Our results provide new insights into pathogenesis of synaptic and neuronal dysfunction in AD.

Exploration of Multistate Conformational Dynamics upon Ligand Binding of a Monomeric Enzyme Involved in Pyrophosphoryl Transfer.

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) is a monomeric protein with 158 residues, which undergoes large-scale conformational changes between apo, open, and holo states responding to ligand binding for its function. It has been explored widely as an excellent target for potential antibacterial drug development. However, little is known about how conformational dynamics between the native states influences the substrate recognition and the functionality of enzymatic catalysis. Here, we report a coarse-grained triple-basin structure-based model upon ligand binding to describe such multiple-state system by the molecular dynamics simulation. With our model, we have made theoretical predictions that are in good agreement with the experimental measurements. Our results revealed the intrinsic conformational fluctuations between apo and open states without ligand binding. We found that HPPK can switch to the activated holo state upon the ordered binding of the two ligands (ATP and HP). We uncovered the underlying mechanism by which major induced fit and minor population shift pathways coexist upon ligand binding by quantitative flux analysis. Additionally, we pointed out the structural origin for the conformational changes and identified the key residues as well as contact interactions. We further explored the temperature effect on the conformational distributions and pathway weights. It gave strong support that higher temperatures promote population shift, while the induced fit pathway is always the predominant activation route of the HPPK system. These findings will provide significant insights of the mechanisms of the multistate conformational dynamics of HPPK upon ligand binding.

Purine-related metabolites and their converting enzymes are altered in frontal, parietal and temporal cortex at early stages of Alzheimer's disease pathology.

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5'-nucleotidase (5'-NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer's disease (AD) and in age-matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5'-NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and ß-amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines.

Thiamine deficiency contributes to synapse and neural circuit defects

BACKGROUND: The previous studies have demonstrated the reduction of thiamine diphosphate is specific to Alzheimer's disease (AD) and causal factor of brain glucose hypometabolism, which is considered as a neurodegenerative index of AD and closely correlates with the degree of cognitive impairment. The reduction of thiamine diphosphate may contribute to the dysfunction of synapses and neural circuits, finally leading to cognitive decline. RESULTS: To demonstrate this hypothesis, we established abnormalities in the glucose metabolism utilizing thiamine deficiency in vitro and in vivo, and we found dramatically reduced dendrite spine density. We further detected lowered excitatory neurotransmission and impaired hippocampal long-term potentiation, which are induced by TPK RNAi in vitro. Importantly, via treatment with benfotiamine, Aß induced spines density decrease was considerably ameliorated. CONCLUSIONS: These results revealed that thiamine deficiency contributed to synaptic dysfunction which strongly related to AD pathogenesis. Our results provide new insights into pathogenesis of synaptic and neuronal dysfunction in AD.

A Network of Conformational Transitions Revealed by Molecular Dynamics Simulations of the Binary Complex of Escherichia coli 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase with MgATP.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the first reaction in the folate biosynthetic pathway. Comparison of its X-ray and nuclear magnetic resonance structures suggests that the enzyme undergoes significant conformational change upon binding to its substrates, especially in three catalytic loops. Experimental research has shown that, in its binary form, even bound by analogues of MgATP, loops 2 and 3 remain rather flexible; this raises questions about the putative large-scale induced-fit conformational change of the HPPK-MgATP binary complex. In this work, long-time all-atomic molecular dynamics simulations were conducted to investigate the loop dynamics in this complex. Our simulations show that, with loop 3 closed, multiple conformations of loop 2, including the open, semiopen, and closed forms, are all accessible to the binary complex. These results provide valuable structural insights into the details of conformational changes upon 6-hydroxymethyl-7,8-dihydropterin (HP) binding and biological activities of HPPK. Conformational network analysis and principal component analysis related to the loops are also discussed.

Structural Basis for the Selective Binding of Inhibitors to 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from Staphylococcus aureus and Escherichia coli.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a member of the folate biosynthesis pathway found in prokaryotes and lower eukaryotes that catalyzes the pyrophosphoryl transfer from the ATP cofactor to a 6-hydroxymethyl-7,8-dihydropterin substrate. We report the chemical synthesis of a series of S-functionalized 8-mercaptoguanine (8MG) analogues as substrate site inhibitors of HPPK and quantify binding against the E. coli and S. aureus enzymes (EcHPPK and SaHPPK). The results demonstrate that analogues incorporating acetophenone-based substituents have comparable affinities for both enzymes. Preferential binding of benzyl-substituted 8MG derivatives to SaHPPK was reconciled when a cryptic pocket unique to SaHPPK was revealed by X-ray crystallography. Differential chemical shift perturbation analysis confirmed this to be a common mode of binding for this series to SaHPPK. One compound (41) displayed binding affinities of 120 nM and 1.76 µM for SaHPPK and EcHPPK, respectively, and represents a lead for the development of more potent and selective inhibitors of SaHPPK.

Molecular dynamics simulations of the Escherichia coli HPPK apo-enzyme reveal a network of conformational transitions.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the first reaction in the folate biosynthetic pathway. Comparison of its X-ray and nuclear magnetic resonance structures suggests that the enzyme undergoes significant conformational change upon binding to its substrates, especially in three catalytic loops. Experimental research has shown that even when confined by crystal contacts, loops 2 and 3 remain rather flexible when the enzyme is in its apo form, raising questions about the putative large-scale induced-fit conformational change of HPPK. To investigate the loop dynamics in a crystal-free environment, we performed conventional molecular dynamics simulations of the apo-enzyme at two different temperatures (300 and 350 K). Our simulations show that the crystallographic B-factors considerably underestimate the loop dynamics; multiple conformations of loops 2 and 3, including the open, semi-open, and closed conformations that an enzyme must adopt throughout its catalytic cycle, are all accessible to the apo-enzyme. These results revise our previous view of the functional mechanism of conformational change upon MgATP binding and offer valuable structural insights into the workings of HPPK. In this paper, conformational network analysis and principal component analysis related to the loops are discussed to support the presented conclusions.

The identification, analysis and structure-based development of novel inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an essential enzyme in the microbial folate biosynthetic pathway. This pathway has proven to be an excellent target for antimicrobial development, but widespread resistance to common therapeutics including the sulfa drugs has stimulated interest in HPPK as an alternative target in the pathway. A screen of a pterin-biased compound set identified several HPPK inhibitors that contain an aryl substituted 8-thioguanine scaffold, and structural analyses showed that these compounds engage the HPPK pterin-binding pocket and an induced cryptic pocket. A preliminary structure activity relationship profile was developed from biophysical and biochemical characterizations of derivative molecules. Also, a similarity search identified additional scaffolds that bind more tightly within the HPPK pterin pocket. These inhibitory scaffolds have the potential for rapid elaboration into novel lead antimicrobial agents.

Exploring the chemical space around 8-mercaptoguanine as a route to new inhibitors of the folate biosynthesis enzyme HPPK.

As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg(2+-)dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (KD ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC50 ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N(9) substitution, or tautomerization arising from N(7) substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N(9)-H···Val46 hydrogen bond, combined with the limited space available around the N(9) position. The water-filled pocket under the N(7) position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N(7) (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (KD ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N(7) position towards the Mg(2+)-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg(2+) centres or Mg(2+)-binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.

Single-molecule photon stamping FRET spectroscopy study of enzymatic conformational dynamics.

The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole-dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: E(FRET) = 1 - τ(DA)/τ(D), where τ(D) and τ(DA) are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy.

Bisubstrate analog inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: new lead exhibits a distinct binding mode.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthesis pathway catalyzing the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin, is an attractive target for developing novel antimicrobial agents. Previously, we studied the mechanism of HPPK action, synthesized bisubstrate analog inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed a new generation of bisubstrate inhibitors by replacing the phosphate bridge with a piperidine-containing linkage. To further improve linker properties, we have synthesized a new compound, characterized its protein binding/inhibiting properties, and determined its structure in complex with HPPK. Surprisingly, this inhibitor exhibits a new binding mode in that the adenine base is flipped when compared to previously reported structures. Furthermore, the side chain of amino acid residue E77 is involved in protein-inhibitor interaction, forming hydrogen bonds with both 2' and 3' hydroxyl groups of the ribose moiety. Residue E77 is conserved among HPPK sequences, but interacts only indirectly with the bound MgATP via water molecules. Never observed before, the E77-ribose interaction is compatible only with the new inhibitor-binding mode. Therefore, this compound represents a new direction for further development.

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor.

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K(d), of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC(50) of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and (15)N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,ß-methylene adenosine 5'-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered--an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including (15)N-(1)H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.

Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their K(d) and IC(50) values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.

Crystal structure of the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase•dihydropteroate synthase bifunctional enzyme from Francisella tularensis.

The 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) enzymes catalyze sequential metabolic reactions in the folate biosynthetic pathway of bacteria and lower eukaryotes. Both enzymes represent validated targets for the development of novel anti-microbial therapies. We report herein that the genes which encode FtHPPK and FtDHPS from the biowarfare agent Francisella tularensis are fused into a single polypeptide. The potential of simultaneously targeting both modules with pterin binding inhibitors prompted us to characterize the molecular details of the multifunctional complex. Our high resolution crystallographic analyses reveal the structural organization between FtHPPK and FtDHPS which are tethered together by a short linker. Additional structural analyses of substrate complexes reveal that the active sites of each module are virtually indistinguishable from those of the monofunctional enzymes. The fused bifunctional enzyme therefore represents an excellent vehicle for finding inhibitors that engage the pterin binding pockets of both modules that have entirely different architectures. To demonstrate that this approach has the potential of producing novel two-hit inhibitors of the folate pathway, we identify and structurally characterize a fragment-like molecule that simultaneously engages both active sites. Our study provides a molecular framework to study the enzyme mechanisms of HPPK and DHPS, and to design novel and much needed therapeutic compounds to treat infectious diseases.

Anharmonic normal mode analysis of elastic network model improves the modeling of atomic fluctuations in protein crystal structures.

Protein conformational dynamics, despite its significant anharmonicity, has been widely explored by normal mode analysis (NMA) based on atomic or coarse-grained potential functions. To account for the anharmonic aspects of protein dynamics, this study proposes, and has performed, an anharmonic NMA (ANMA) based on the C(alpha)-only elastic network models, which assume elastic interactions between pairs of residues whose C(alpha) atoms or heavy atoms are within a cutoff distance. The key step of ANMA is to sample an anharmonic potential function along the directions of eigenvectors of the lowest normal modes to determine the mean-squared fluctuations along these directions. ANMA was evaluated based on the modeling of anisotropic displacement parameters (ADPs) from a list of 83 high-resolution protein crystal structures. Significant improvement was found in the modeling of ADPs by ANMA compared with standard NMA. Further improvement in the modeling of ADPs is attained if the interactions between a protein and its crystalline environment are taken into account. In addition, this study has determined the optimal cutoff distances for ADP modeling based on elastic network models, and these agree well with the peaks of the statistical distributions of distances between C(alpha) atoms or heavy atoms derived from a large set of protein crystal structures.

Probing the roles of non-homologous insertions in the N-terminal domain of Plasmodium falciparum hydroxymethylpterin pyrophosphokinase-dihydropteroate synthase.

Plasmodium falciparum bifunctional hydroxymethylpterin pyrophosphokinase-dihydropteroate synthase (pfHPPK-DHPS) is a crucial enzyme in the de novo folate biosynthesis pathway. The crystal structure is not yet available for this enzyme, however, homology model of the enzyme reported previously revealed the presence of parasite-specific insertions. Alignment of pfHPPK-DHPS with HPPK and DHPS sequences from other microorganisms reveals two insertions relative to the corresponding enzyme in other organisms, i.e. HPPK-1 and HPPK-2. The former encompasses amino acid residues 66-162, while the latter covers residues 213-311. In order to investigate the roles of the two insertions, we constructed a number of mutants in which parts of these two insertions were deleted. Characterization of the mutationally altered proteins revealed that deletions of residues 74-80 in the HPPK-1 sequence of the pfHPPK-DHPS, but not that of the monofunctional pfHPPK, decreased the HPPK activity. A longer deletion (residues 74-86) in the HPPK-1 sequence of the bifunctional pfHPPK-DHPS completely inactivated both HPPK and DHPS activities. However, deletion in the HPPK-2 sequence from residues 247-306 did not disrupt the activities of HPPK and DHPS, but the kinetic properties of the recombinant proteins were slightly changed. The importance of HPPK-1 sequence on the catalytic activities of HPPK and DHPS in the bifunctional pfHPPK-DHPS could have implications for development of inhibitors targeting the non-catalytic region of this chemotherapeutically important enzyme.

Dynamics of the conformational transitions in the assembling of the Michaelis complex of a bisubstrate enzyme: a (15)N relaxation study of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), which follows an ordered bi-bi kinetic mechanism with ATP binding to the enzyme first. HPPK undergoes dramatic conformational changes during its catalytic cycle as revealed by X-ray crystallography, and the conformational changes are essential for the enzymatic catalysis as shown by site-directed mutagenesis and biochemical and crystallographic analysis of the mutants. However, the dynamic properties of the enzyme have not been measured experimentally. Here, we report a (15)N NMR relaxation study of the dynamic properties of Escherichia coli HPPK from the apo form to the binary substrate complex with MgATP (represented by MgAMPCPP, an ATP analogue) to the Michaelis complex (ternary substrate complex) with MgATP (represented by MgAMPCPP) and HP (represented by 7,7-dimethyl-6-hydroxypterin, an HP analogue). The results show that the binding of the nucleotide to HPPK does not cause major changes in the dynamic properties of the enzyme. Whereas enzymes are often more rigid when bound to the ligand or the substrate, the internal mobility of HPPK is not reduced and is even moderately increased in the binary complex, particularly in the catalytic loops. The internal mobility of the catalytic loops is significantly quenched upon the formation of the ternary complex, but some mobility remains. The enhanced motions in the catalytic loops of the binary substrate complex may be required for the assembling of the ternary complex. On the other hand, some degrees of mobility in the catalytic loops of the ternary complex may be required for the optimal stabilization of the transition state, which may need the instantaneous adjustment and alignment of the side-chain positions of catalytic residues. Such dynamic behaviors may be characteristic of bisubstrate enzymes.

The structure and mechanism of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyses the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), and is an essential enzyme in the biosynthesis of folic acid. It is also a potential target for antimicrobial drugs. HPPK from Escherichia coli, which has been the most intensively investigated, is a monomeric protein with a molecular mass of about 18,000. Structures of the enzyme, determined by X-ray crystallography and NMR, have shown that it adopts an alpha/beta fold with a substrate-binding cleft on the surface. Three loop regions surround the enzyme active site and form intimate contacts with the substrates. The enzyme has a fixed order of substrate binding, with ATP binding first, followed by HMDP. Binding of ATP causes a shift in the conformations of the loop regions, which completes formation of the HMDP-binding site. Two magnesium ions bind within the active site, bridging between the phosphate groups in ATP and the enzyme. Both ions appear to play an integral role in ATP recognition and stabilization of the transition state of the reaction. Ligand binding and kinetic studies have shown that the overall rate of the reaction is not limited by the rate of substrate transformation into products on the enzyme, which is relatively fast, but is more likely caused by a slow step associated with product release. These fundamental studies open up the potential for exploitation through the design of specific HPPK inhibitors.

Sampling small-scale and large-scale conformational changes in proteins and molecular complexes.

Sampling of small-scale and large-scale motions is important in various computational tasks, such as protein-protein docking and ligand binding. Here, we report further development and applications of the activation-relaxation technique for internal coordinate space trajectories (ARTIST). This method generates conformational moves of any complexity and size by identifying and crossing well-defined saddle points connecting energy minima. Simulations on two all-atom proteins and three protein complexes containing between 70 and 300 amino acids indicate that ARTIST opens the door to the full treatment of all degrees of freedom in dense systems such as protein-protein complexes.