Interdomain compactization in human tyrosyl-tRNA synthetase studied by the hierarchical rotations technique

Aminoacyl-tRNA synthetases are key enzymes of protein biosynthesis which usually possess multidomain structures. Mammalian tyrosyl-tRNA synthetase is composed of two structural modules: N-terminal catalytic core and an EMAPII-like C-terminal domain separated by long flexible linker. The structure of full-length human cytoplasmic tyrosyl-tRNA synthetase is still unknown. The structures of isolated N-terminal and C-terminal domains of the protein are resolved, but their compact packing in a functional enzyme is a subject of debates. In this work we studied putative compactization of the N- and C-terminal modules of human tyrosyl-tRNA synthetase by the coarse-grained hierarchical rotations technique (HIEROT). The large number of distinct types of binding interfaces between N- and C-terminal modules is revealed in the absence of enzyme substrates. The binding propensities of different residues are computed and several binding "hot spots" are observed on the surfaces of N and C modules. These results could be used to govern atomistic molecular dynamics simulations, which will sample preferable binding interfaces effectively.     

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors

The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.     

Correlations between kinetic and X-ray analyses of engineered enzymes: crystal structures of mutants Cys----Gly-35 and Tyr----Phe-34 of tyrosyl-tRNA synthetase

The crystal structures of two mutant tyrosyl-tRNA synthetases (TyrTS) are reported to test predictions from kinetic data about structural perturbations and also to aid in the interpretation of apparent strengths of hydrogen bonds measured by protein engineering. The enzyme-tyrosine and enzyme-tyrosyl adenylate complexes of the mutant, TyrTS(Cys----Gly-35), have been determined at 2.5- and 2.7-A resolution, respectively. Residue Cys-35 is in the ribose binding site. Small rearrangements in structure are seen in the enzyme-tyrosine complex that are localized around the cavity created by the mutation. The side chain of Thr-51 moves to occupy the cavity, and Ile-52 adopts two significantly populated conformations, one as in the native enzyme and a second unique to the mutant. On binding tyrosyl adenylate, Ile-52 in the mutant crystal structure preferentially occupies the conformation observed in the native structure. The side chain at Thr-51 becomes disordered. The double-mutant test, which was designed to detect interactions between residues, had previously shown a discrepancy of some 0.4 kcal/mol on mutating Cys-35 and Thr-51 separately and together. A crystal structure of a second mutant, delta TyrTS(Tyr----Phe-34), complexed with tyrosine has been determined at 2.7-A resolution. Tyr-34 in wild-type enzyme makes a hydrogen bond with the phenolic oxygen of the bound tyrosine substrate. The mutant crystal structure was solved to discover whether or not a water molecule binds to the substrate instead of the hydroxyl of Tyr-34 as the interpretation of apparent binding energies from site-directed mutagenesis experiments hinges crucially on whether there is access of water to the mutated region.     

Natural variation of tyrosyl-tRNA synthetase and comparison with engineered mutants

We report the cloning and sequence analysis of the gene for the tyrosyl-tRNA synthetase from Bacillus caldotenax and properties of the gene product. The amino acid sequence of the tyrosyl-tRNA synthetase was found to be 99% homologous with the corresponding enzyme from B. stearothermophilus, with only four amino acid differences. Two of these natural variations were found to involve active site residues of the enzyme and correspond to mutations that have been engineered previously in vitro. One, Thr-51----Ala-51, produced a more active enzyme, possessing a higher value of kcat/KM for ATP. Position 51 is a "hot spot" in the tyrosyl-tRNA synthetase, differing in enzymes derived from Escherichia coli, B. stearothermophilus, and B. caldotenax. The other, His-48----Asn-48, is found to be a neutral mutation but is in one of the rare regions that are conserved with other aminoacyl-tRNA synthetases. The equivalence of histidine and asparagine at position 48 extends the homology in this region to more enzymes. These residues, His-Ile-Gly-His, and now His-Ile-Gly-Asn, form part of the binding site for ATP in the transition state of the reaction. Although B. caldotenax is an obligate thermophile with an optimal growth temperature of 80 degrees C, as much as 20 degrees C above the growth optima of strains of Bacillus stearothermophilus, its tyrosyl-tRNA synthetase has an identical thermal stability in vitro to that from B. stearothermophilus.     

Structure of a closed form of human malic enzyme and implications for catalytic mechanism

Malic enzymes are widely distributed in nature and have many biological functions. The crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme in a quaternary complex with NAD+, Mn++ and oxalate has been determined at 2.2 A resolution. The structures of the quaternary complex with NAD+, Mg++, tartronate or ketomalonate have been determined at 2.6 A resolution. The structures show the enzyme in a closed form in these complexes and reveal the binding modes of the cation and the inhibitors. The divalent cation is coordinated in an octahedral fashion by six ligating oxygens, two from the substrate/inhibitor, three from Glu 255, Asp 256 and Asp 279 of the enzyme, and one from a water molecule. The structural information has significant implications for the catalytic mechanism of malic enzymes and identifies Tyr 112 and Lys 183 as possible catalytic residues. Changes in tetramer organization of the enzyme are also observed in these complexes, which might be relevant for its cooperative behavior and allosteric control.     

Interaction of tyrosyl aryl dipeptides with S. aureus tyrosyl tRNA synthetase: inhibition and crystal structure of a complex.

Tyrosyl aryl dipeptide inhibitors of S. aureus tyrosyl tRNA synthetase have been identified with IC50 values down to 0.5 microM. A crystal structure of the enzyme complexed to one of the inhibitors shows occupancy of the tyrosyl binding pocket coupled with inhibitor interactions to key catalytic residues.     

Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.     

Crystal structure of type II peptide deformylase from Staphylococcus aureus

The first crystal structure of Class II peptide deformylase has been determined. The enzyme from Staphylococcus aureus has been overexpressed and purified in Escherichia coli and the structure determined by x-ray crystallography to 1.9 A resolution. The purified iron-enriched form of S. aureus peptide deformylase enzyme retained high activity over many months. In contrast, the iron-enriched form of the E. coli enzyme is very labile. Comparison of the two structures details many differences; however, there is no structural explanation for the dramatic activity differences we observed. The protein structure of the S. aureus enzyme reveals a fold similar, but not identical to, the well characterized E. coli enzyme. The most striking deviation of the S. aureus from the E. coli structure is the unique conformation of the C-terminal amino acids. The distinctive C-terminal helix of the latter is replaced by a strand in S. aureus which wraps around the enzyme, terminating near the active site. Although there are no differences at the amino acid level near the active site metal ion, significant changes are noted in the peptide binding cleft which may play a role in the design of general peptide deformylase inhibitors.     

X-ray structure of 4,4'-dihydroxybenzophenone mimicking sterol substrate in the active site of sterol 14alpha-demethylase (CYP51)

A universal step in the biosynthesis of membrane sterols and steroid hormones is the oxidative removal of the 14alpha-methyl group from sterol precursors by sterol 14alpha-demethylase (CYP51). This enzyme is a primary target in treatment of fungal infections in organisms ranging from humans to plants, and development of more potent and selective CYP51 inhibitors is an important biological objective. Our continuing interest in structural aspects of substrate and inhibitor recognition in CYP51 led us to determine (to a resolution of 1.95A) the structure of CYP51 from Mycobacterium tuberculosis (CYP51(Mt)) co-crystallized with 4,4'-dihydroxybenzophenone (DHBP), a small organic molecule previously identified among top type I binding hits in a library screened against CYP51(Mt). The newly determined CYP51(Mt)-DHBP structure is the most complete to date and is an improved template for three-dimensional modeling of CYP51 enzymes from fungal and prokaryotic pathogens. The structure demonstrates the induction of conformational fit of the flexible protein regions and the interactions of conserved Phe-89 essential for both fungal drug resistance and catalytic function, which were obscure in the previously characterized CYP51(Mt)-estriol complex. DHBP represents a benzophenone scaffold binding in the CYP51 active site via a type I mechanism, suggesting (i) a possible new class of CYP51 inhibitors targeting flexible regions, (ii) an alternative catalytic function for bacterial CYP51 enzymes, and (iii) a potential for hydroxybenzophenones, widely distributed in the environment, to interfere with sterol biosynthesis. Finally, we show the inhibition of M. tuberculosis growth by DHBP in a mouse macrophage model.     

The 'KMSKS' motif in tyrosyl-tRNA synthetase participates in the initial binding of tRNA(Tyr)

Variants at each position of the 'KMSKS' signature motif in tyrosyl-tRNA synthetase have been analyzed to test the hypothesis that this motif is involved in catalysis of the second step of the aminoacylation reaction (i.e., the transfer of tyrosine from the enzyme-bound tyrosyl-adenylate intermediate to the tRNA(Tyr) substrate). Pre-steady-state kinetic studies show that while the rate constants for tyrosine transfer (k(4)) are similar to the wild-type value for all of the mobile loop variants, the K230A and K233A variants have increased dissociation constants (K(d)(tRNA)( )()= 2.4 and 1.7 microM, respectively) relative to the wild-type enzyme (K(d)(tRNA)( )()= 0.39 microM). In contrast, the K(d)(tRNA) values for the F231L, G232A, and T234A variants are similar to that of the wild-type enzyme. The K(d)(tRNA) value for a loop deletion variant, Delta(227-234), is similar to that for the K230A/K233A double mutant variant (3.4 and 3.0 microM, respectively). Double mutant free energy cycle analysis indicates there is a synergistic interaction between the side chains of K230 and K233 during the initial binding of tRNA(Tyr) (DeltaDeltaG(int) = -0.74 kcal/mol). These results suggest that while the 'KMSKS' motif is important for the initial binding of tRNA(Tyr) to tyrosyl-tRNA synthetase, it does not play a catalytic role in the second step of the reaction. These studies provide the first kinetic evidence that the 'KMSKS' motif plays a role in the initial binding of tRNA(Tyr) to tyrosyl-tRNA synthetase.     

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gamma phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.     

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gi phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.     

Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 A), sulfometuron methyl (2.34 A), and two other sulfonylureas, metsulfuron methyl (2.29 A) and tribenuron methyl (2.58 A). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.     

Crystal structure of lumazine synthase from Mycobacterium tuberculosis as a target for rational drug design: binding mode of a new class of purinetrione inhibitors

The enzymes involved in the biosynthesis of riboflavin represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with enzymes of the mammalian metabolism. Lumazine synthase catalyzes the penultimate step in the riboflavin biosynthesis pathway. A number of substituted purinetrione compounds represent a new class of highly specific inhibitors of lumazine synthase from Mycobacterium tuberculosis. To develop potent antibiotics for the treatment of tuberculosis, we have determined the structure of lumazine synthase from M. tuberculosis in complex with two purinetrione inhibitors and have studied binding via isothermal titration calorimetry. The structures were determined by molecular replacement using lumazine synthase from Saccharomyces cerevisiae as a search model and refined at 2 and 2.3 A resolution. The R-factors were 14.7 and 17.4%, respectively, and the R(free) values were 19.3 and 26.3%, respectively. The enzyme was found to be a pentamer consisting of five subunits related by 5-fold local symmetry. The comparison of the active site architecture with the active site of previously determined lumazine synthase structures reveals a largely conserved topology with the exception of residues Gln141 and Glu136, which participate in different charge-charge interactions in the core space of the active site. The impact of structural changes in the active site on the altered binding and catalytic properties of the enzyme is discussed. Isothermal titration calorimetry measurements indicate highly specific binding of the purinetrione inhibitors to the M. tuberculosis enzyme with dissociation constants in micromolar range.     

Structural and biochemical studies of inhibitor binding to human cytomegalovirus protease

Herpesvirus protease is required for the life cycle of the virus and is an attractive target for the design and development of new anti-herpes agents. The protease belongs to a new class of serine proteases, with a novel backbone fold and a unique Ser-His-His catalytic triad. Here we report the crystal structures of human cytomegalovirus protease in complex with two peptidomimetic inhibitors. The structures reveal a new hydrogen-bonding interaction between the main chain carbonyl of the P(5) residue and the main chain amide of amino acid 137 of the protease, which is important for the binding affinity of the inhibitor. Conformational flexibility was observed in the S(3) pocket of the enzyme, and this is supported by our characterization of several mutants in this pocket. One of the structures is at 2.5 A resolution, allowing us for the first time to locate ordered solvent molecules in the inhibitor complex. The presence of two solvent molecules in the active site may have implications for the design of new inhibitors against this enzyme. Favorable and stereospecific interactions have been established in the S(1)' pocket for one of these inhibitors.     

Crystal structure and substrate specificity of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

beta-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 A resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- > acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.     

The structure of CYP101D2 unveils a potential path for substrate entry into the active site

We describe in the present paper mutations of the catalytic subunit α of PKA (protein kinase A) that introduce amino acid side chains into the ATP-binding site and progressively transform the pocket to mimic that of Aurora protein kinases. The resultant PKA variants are enzymatically active and exhibit high affinity for ATP site inhibitors that are specific for Aurora kinases. These features make the Aurora-chimaeric PKA a valuable tool for structure-based drug discovery tasks. Analysis of crystal structures of the chimaera reveal the roles for individual amino acid residues in the binding of a variety of inhibitors, offering key insights into selectivity mechanisms. Furthermore, the high affinity for Aurora kinase-specific inhibitors, combined with the favourable crystallizability properties of PKA, allow rapid determination of inhibitor complex structures at an atomic resolution. We demonstrate the utility of the Aurora-chimaeric PKA by measuring binding kinetics for three Aurora kinase-specific inhibitors, and present the X-ray structures of the chimaeric enzyme in complex with VX-680 (MK-0457) and JNJ-7706621 [Aurora kinase/CDK (cyclin-dependent kinase) inhibitor].     

A novel NAD-binding protein revealed by the crystal structure of 2,3-diketo-L-gulonate reductase (YiaK)

Escherichia coli YiaK catalyzes the reduction of 2,3-diketo-L-gulonate in the presence of NADH. It belongs to a large family of oxidoreductases that is conserved in archaea, bacteria, and eukaryotes but shows no sequence homology to other proteins. We report here the crystal structures at up to 2.0-A resolution of YiaK alone and in complex with NAD-tartrate. YiaK has a new polypeptide backbone fold and a novel mode of recognizing the NAD cofactor. In addition, NAD is bound in an unusual conformation, at the interface of a dimer of the enzyme. The crystallographic analysis unexpectedly revealed the binding of tartrate in the active site. Enzyme kinetics studies confirm that tartrate and the related D-malate are inhibitors of YiaK. In contrast to most other enzymes where substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site. The free enzyme conformation is incompatible with NAD binding. His(44) is likely the catalytic residue of the enzyme.     

Secondary structure of the C-terminal domain of the tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus: a novel type of anticodon binding domain?

The tyrosyl-tRNA synthetase catalyzes the activation of tyrosine and its coupling to the cognate tRNA. The enzyme is made of two domains: an N-terminal catalytic domain and a C-terminal domain that is necessary for tRNA binding and for which it was not possible to determine the structure by X-ray crystallography. We determined the secondary structure of the C-terminal domain of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus by nuclear magnetic resonance methods and found that it is of the alpha+beta type. Its arrangement differs from those of the other anticodon binding domains whose structure is known. We also found that the isolated C-terminal domain behaves as a folded globular protein, and we suggest the presence of a flexible linker between the two domains.     

A structural variation for MurB: X-ray crystal structure of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB)

The X-ray crystal structure of the substrate free form of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB) has been solved to 2.3 A resolution with an R-factor of 20.3% and a free R-factor of 22.3%. While the overall fold of the S. aureus enzyme is similar to that of the homologous Escherichia coli MurB X-ray crystal structure, notable distinctions between the S. aureus and E. coli MurB protein structures occur in residues involved in substrate binding. Analysis of available MurB sequences from other bacteria suggest that the S. aureus MurB structure is representative of a distinct structural class of UDP-N-acetylenolpyruvylglucosamine reductases including Bacillus subtilis and Helicobacter pylori that are characterized by a modified mechanism for substrate binding.