Rat submaxillary gland serine protease, tonin. Structure solution and refinement at 1.8 A resolution

Tonin is a mammalian serine protease that is capable of generating the vasoconstrictive agent, angiotensin II, directly from its precursor protein, angiotensinogen, a process that normally requires two enzymes, renin and angiotensin-converting enzyme. The X-ray crystallographic structure determination and refinement of tonin at 1.8 A resolution and the analysis of the resulting model are reported. The initial phases were obtained by the method of molecular replacement using as the search model the structure of bovine trypsin. The refined model of tonin consists of 227 amino acid residues out of the 235 in the complete molecule, 149 water molecules, and one zinc ion. The R-factor (R = sigma Fo - Fc/sigma Fo) is 0.196 for the 14,997 measured data between 8 and 1.8 A resolution with I greater than or equal to sigma (I). It is estimated that the overall root-mean-square error in the coordinates is about 0.3 A. The structure of tonin that has been determined is not in its active conformation, but one that has been perturbed by the binding of Zn2+ in the active site. Zn2+ was included in the buffer to aid the crystallization. Nevertheless, the structure of tonin that is described is for the most part similar to its native form as indicated by the close tertiary structural homology with kallikrein. The differences in the structures of the two enzymes are concentrated in several loop regions; these structural differences are probably responsible for the differences in their reactivities and specificities.     

Structure of a pancreatic alpha-amylase bound to a substrate analogue at 2.03 A resolution.

The structure of pig pancreatic alpha-amylase in complex with carbohydrate inhibitor and proteinaceous inhibitors is known but the successive events occurring at the catalytic center still remain to be elucidated. The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) soaked with an enzyme-resistant substrate analogue, methyl 4,4'-dithio-alpha-maltotrioside, showed electron density corresponding to the binding of substrate analogue molecules at the active site and at the "second binding site." The electron density observed at the active site was interpreted in terms of overlapping networks of oligosaccharides, which show binding of substrate analogue molecules at subsites prior to and subsequent to the cleavage site. A weaker patch of density observed at subsite -1 (using a nomenclature where the site of hydrolysis is taken to be between subsites -1 and +1) was modeled with water molecules. Conformational changes take place upon substrate analogue binding and the "flexible loop" that constitutes the surface edge of the active site is observed in a specific conformation. This confirms that this loop plays an important role in the recognition and binding of the ligand. The crystal structure was refined at 2.03 A resolution, to an R-factor of 16.0 (Rfree, 18.5).     

The atomic structure of pentameric lumazine synthase from Saccharomyces cerevisiae at 1.85 A resolution reveals the binding mode of a phosphonate intermediate analogue

Lumazine synthase of Saccharomyces cerevisiae is a homopentamer with a molecular weight of 90 kDa. Crystals of the recombinant enzyme with a size of up to 1.6 mm were obtained. The space group is P4(1)2(1)2 with lattice dimensions 82.9 A x 82.9 A x 300.2 A. X-ray diffraction data collected under cryogenic conditions were complete to 1.85 A resolution. The structure of the enzyme in complex with the intermediate analogue, 5-(6-D-ribitylamino-2,4-dihydroxypyrimidine-5-yl)-1-pentyl-p hosphonic acid was solved via molecular replacement using the structure of the Bacillus subtilis enzyme as search model and was refined to a final R-factor of 19.8% (Rfree: 22.5%). The conformation of the active site ligand of the enzyme mimicks that of the Schiff base intermediate of the enzyme-catalyzed reaction. The data enable the reconstruction of the reactant topology during the early steps of the catalytic reaction. Structural determinants, which are likely to be responsible for the inability of the S. cerevisiae enzyme to form icosahedral capsids, will be discussed.     

Refinement of an enzyme complex with inhibitor bound at partial occupancy. Hen egg-white lysozyme and tri-N-acetylchitotriose at 1.75 A resolution.

The structure of the tri-N-acetylchitotriose inhibitor complex of hen egg-white lysozyme has been refined at 1.75 A resolution, using data collected from a complex crystal with ligand bound at less than full occupancy. To determine the exact value of the inhibitor occupancy, a model comprising unliganded and sugar-bound protein molecules was generated and refined against the 1.75 A data, using a modified version of the Hendrickson & Konnert least-squares procedure. The crystallographic R-factor for the model was found to fall to a minimum at 55% bound sugar. Conventional refinement assuming unit occupancy was found to yield incorrect thermal and positional parameters. Application of the same refinement procedures to an earlier 2.0 A data set, collected independently on different complex crystals by Blake et al. gave less consistent results than the 1.75 A refinement. From an analysis of the high resolution structure a detailed picture of the protein-carbohydrate interactions in the non-productive complex has emerged, together with the conformation and mobility changes that accompany ligand binding. The specificity of interaction between the protein and inhibitor, bound in subsites A to C of the active site, is seen to be generated primarily by an extensive network of hydrogen bonds, both to the protein itself and to bound solvent molecules. The latter also play an important role in maintaining the structural integrity of the active site cleft in the apo-protein.     

Three-dimensional structures of glycolate oxidase with bound active-site inhibitors.

A key step in plant photorespiration, the oxidation of glycolate to glyoxylate, is carried out by the peroxisomal flavoprotein glycolate oxidase (EC 1.1.3.15). The three-dimensional structure of this alpha/beta barrel protein has been refined to 2 A resolution (Lindqvist Y. 1989. J Mol Biol 209:151-166). FMN dependent glycolate oxidase is a member of the family of alpha-hydroxy acid oxidases. Here we describe the crystallization and structure determination of two inhibitor complexes of the enzyme, TKP (3-Decyl-2,5-dioxo-4-hydroxy-3-pyrroline) and TACA (4-Carboxy-5-(1-pentyl)hexylsulfanyl-1,2,3-triazole). The structure of the TACA complex has been refined to 2.6 A resolution and the TKP complex, solved with molecular replacement, to 2.2 A resolution. The Rfree for the TACA and TKP complexes are 24.2 and 25.1%, respectively. The overall structures are very similar to the unliganded holoenzyme, but a closer examination of the active site reveals differences in the positioning of the flavin isoalloxazine ring and a displaced flexible loop in the TKP complex. The two inhibitors differ in binding mode and hydrophobic interactions, and these differences are reflected by the very different Ki values for the inhibitors, 16 nM for TACA and 4.8 microM for TKP. Implications of the structures of these enzyme-inhibitor complexes for the model for substrate binding and catalysis proposed from the holo-enzyme structure are discussed.     

Crystallographic analysis of the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae identifies citrate as a ligand to the molybdenum of iron molybdenum cofactor (FeMoco)

The x-ray crystal structure of NifV(-) Klebsiella pneumoniae nitrogenase MoFe protein (NifV(-) Kp1) has been determined and refined to a resolution of 1.9 A. This is the first structure for a nitrogenase MoFe protein with an altered cofactor. Moreover, it is the first direct evidence that the organic acid citrate is not just present, but replaces homocitrate as a ligand to the molybdenum atom of the iron molybdenum cofactor (FeMoco). Subsequent refinement of the structure revealed that the citrate was present at reduced occupancy.     

Further refinement of the structure of yeast tRNAPhe.

We have refined the monoclinic crystal structure of yeast tRNA^Phe against a complete set of X-ray data at 2.5 Å resolution, using real-space refinement and a combination of energy minimisation and crystallographic least-squares. This refinement has allowed us to define the conformation of residue D16, and to make corrections to Y37 and A76. We have found an additional magnesium binding site (making a total of four), a number of water molecules, and a possible spermine molecule.A revised list of torsion angles is given.     

Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.

The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D

The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D.     

Crystal structure of thermitase at 1.4 A resolution

The crystal structure of thermitase, a subtilisin-type serine proteinase from Thermoactinomyces vulgaris, was determined by X-ray diffraction at 1.4 A resolution. The structure was solved by a combination of molecular and isomorphous replacement. The starting model was that of subtilisin BPN' from the Protein Data Bank, determined at 2.5 A resolution. The high-resolution refinement was based on data collected using synchrotron radiation with a Fuji image plate as detector. The model of thermitase refined to a conventional R factor of 14.9% and contains 1997 protein atoms, 182 water molecules and two Ca ions. The tertiary structure of thermitase is similar to that of the other subtilisins although there are some significant differences in detail. Comparison with subtilisin BPN' revealed two major structural differences. The N-terminal region in thermitase, which is absent in subtilisin BPN', forms a number of contacts with the tight Ca2+ binding site and indeed provides the very tight binding of the Ca ion. In thermitase the loop of residues 60 to 65 forms an additional (10) beta-strand of the central beta-sheet and the second Ca2+ binding site that has no equivalent in the subtilisin BPN' structure. The observed differences in the Ca2+ binding and the increased number of ionic and aromatic interactions in thermitase are likely sources of the enhanced stability of thermitase.     

Homology model of the CDK1/cyclin B complex

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.     

Refined 1.8 A crystal structure of the lambda repressor-operator complex.

The crystal structure of the lambda repressor-operator complex has been refined to an R-factor of 18.9% at 1.8 A resolution. This refinement, using data collected at low temperature, has revealed the structure of the N-terminal arm and shows that the interactions of repressor with the two halves of the pseudo-symmetric operator site are significantly different. The two halves of the complex are most similar near the outer edge of the operator site (in a region where the lambda and 434 repressors make similar contacts), but they become increasingly different toward the center of the operator. There are striking differences near the center of the site where it appears that the arm makes significant contacts to only one half of the DNA site. This suggested a new way of aligning the operator sites in phage lambda. The high resolution structure confirms many of the previously noted features of the complex, but also reveals a number of new protein-DNA contacts. It also gives a better view of the extensive H-bonding networks that couple contacts made by different residues and different regions of the protein, and reveals important new details about the helix-turn-helix (HTH) region, and the positions of many water molecules in the complex.     

Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A resolution: comparison of the unligated enzyme and a complex with alpha-methyl-l-glutamate

Phosphoserine aminotransferase (PSAT; EC 2.6.1.52), a member of subgroup IV of the aminotransferases, catalyses the conversion of 3-phosphohydroxypyruvate to l-phosphoserine. The crystal structure of PSAT from Escherichia coli has been solved in space group P212121 using MIRAS phases in combination with density modification and was refined to an R-factor of 17.5% (Rfree=20.1 %) at 2.3 A resolution. In addition, the structure of PSAT in complex with alpha-methyl-l-glutamate (AMG) has been refined to an R-factor of 18.5% (Rfree=25.1%) at 2.8 A resolution. Each subunit (361 residues) of the PSAT homodimer is composed of a large pyridoxal-5'-phosphate binding domain (residues 16-268), consisting of a seven-stranded mainly parallel beta-sheet, two additional beta-strands and seven alpha-helices, and a small C-terminal domain, which incorporates a five-stranded beta-sheet and two alpha-helices. A three-dimensional structural comparison to four other vitamin B6-dependent enzymes reveals that three alpha-helices of the large domain, as well as an N-terminal domain (subgroup II) or subdomain (subgroup I) are absent in PSAT. Its only 15 N-terminal residues form a single beta-strand, which participates in the beta-sheet of the C-terminal domain. The cofactor is bound through an aldimine linkage to Lys198 in the active site. In the PSAT-AMG complex Ser9 and Arg335 bind the AMG alpha-carboxylate group while His41, Arg42 and His328 are involved in binding the AMG side-chain. Arg77 binds the AMG side-chain indirectly through a solvent molecule and is expected to position itself during catalysis between the PLP phosphate group and the substrate side-chain. Comparison of the active sites of PSAT and aspartate aminotransferase suggests a similar catalytic mechanism, except for the transaldimination step, since in PSAT the Schiff base is protonated. Correlation of the PSAT crystal structure to a published profile sequence analysis of all subgroup IV members allows active site modelling of nifs and the proposal of a likely molecular reaction mechanism.     

X-ray structural analysis of magnesium-containing Serratia marcescens endonuclease]

The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescens was refined at the resolution of 1.07 A to R factor of 12.4% and Rfree factor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg2+ ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N- and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg2+ ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O delta 1 atom of Asn119 were shown to be within a range of 2.01-2.11 A. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 A2, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 A2, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.     

Structure and function of chromophores in R-Phycoerythrin at 1.9 A resolution

The crystal structure of R-Phycoerythrin (R-PE) from Polysiphonia urceolata has been refined to a resolution of 1.9 A, based on the atomic coordinates of R-PE determined at 2.8 A resolution, through the use of difference Fourier method and steorochemistry parameters restrained refinement with model adjustment according to the electron density map. Crystallographic R-factor of the refined model is 0.195 (Rfree = 0.282) from 8-1.9 A. High resolution structure of R-PE showed precise interactions between the chromophores and protein residues, which explained the spectrum characteristic and function of chromophores. Four chiral atoms of phycourobilin (PUB) were identified as C(4)-S, C(16)-S, C(21)-S, and C(20)-R. In addition to the coupling distances of 19 A to 45 A between the chromophores which were observed and involved in the energy transfer pathway, high resolution structure of R-PE suggested other pathways of energy transfer, such as the ultrashort distance between alpha140a and beta155. It has been proposed that aromatic residues in linker proteins not only influence the conformation of chromophore, but may also bridge chromophores to improve the energy transfer efficiency.     

The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43, with a C-terminal beta-barrel domain

The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43 from Bacillus sp. KSM-KP43, with a C-terminal extension domain, was determined by the multiple isomorphous replacements method with anomalous scattering. The native form was refined to a crystallographic R factor of 0.134 (Rfree of 0.169) at 1.30-A resolution. KP-43 consists of two domains, a subtilisin-like alpha/beta domain and a C-terminal jelly roll beta-barrel domain. The topological architecture of the molecule is similar to that of kexin and furin, which belong to the subtilisin-like proprotein convertases, whereas the amino acid sequence and the binding orientation of the C-terminal beta-barrel domain both differ in each case. Since the C-terminal domains of subtilisin-like proprotein convertases are essential for folding themselves, the domain of KP-43 is also thought to play such a role. KP-43 is known to be an oxidation-resistant protease among the general subtilisin-like proteases. To investigate how KP-43 resists oxidizing reagents, the structure of oxidized KP-43 was also determined and refined to a crystallographic R factor of 0.142 (Rfree of 0.212) at 1.73-A resolution. The structure analysis revealed that Met-256, adjacent to catalytic Ser-255, was oxidized similarly to an equivalent residue in subtilisin BPN'. Although KP-43, as well as proteinase K and subtilisin Carlsberg, lose their hydrolyzing activity against synthetic peptides after oxidation treatment, all of them retain 70-80% activity against proteinaceous substrates. These results, as well as the beta-casein digestion pattern analysis, have indicated that the oxidation of the methionine adjacent to the catalytic serine is not a dominant modification but might alter the substrate specificities.     

Homology Model of the CDK1/cyclin B Complex

Abstract

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.     

Comparison of binding interactions of dibromoflavonoids with transthyretin

The crystal structure of rat transthyretin (rTTR) complex with the dibromoflavone EMD21388 was determined to 2.3 A resolution and refined to R = 0.203 and Rfree = 0.288. Two different orientations of EMD21388, which differ in the channel penetration by 1.6 A, were found in the A/C binding site of rTTR. The single ligand position observed in the BID site is intermediate between the two positions found in the A/C site. The position of the dibromoflavone in the B/D site is similar to that reported for dibromoaurone in human TTR. The bromine atoms of EMD21388 form strong interactions in the P3 and P3' pockets of rTTR. Due to the different molecular architectures of both ligands, dibromoflavone forms only one interaction with Lys-15 near the channel entrance, while direct interactions with the pair of Lys-15 were reported for dibromoaurone. The C3* methyl group of EMD21388 mediates the bridging interactions between two TTR subunits in the P2 pockets. The interactions of the O2* hydroxyl group of dibromoaurone with the Thr-119 side chain in the P3 pockets are not matched by similar interactions in EMD21388. Both these alternative interactions can explain the competitive binding of 3',5'-dibromoflavonoids to transthyretin.     

Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution

The structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus has been crystallographically refined at 1.8 A resolution using restrained least-squares refinement methods. The final crystallographic R-factor for 93,120 reflexions with F greater than 3 sigma (F) is 0.177. The asymmetric unit of the crystal contains a complete tetramer, the final model of which incorporates a total of 10,272 unique protein and coenzyme atoms together with 677 bound solvent molecules. The structure has been analysed with respect to molecular symmetry, intersubunit contacts, coenzyme binding and active site geometry. The refined model shows the four independent subunits to be remarkable similar apart from local deviations due to intermolecular contacts within the crystal lattice. A number of features are revealed that had previously been misinterpreted from an earlier 2.7 A electron density map. Arginine at position 195 (previously thought to be a glycine) contributes to the formation of the anion binding sites in the active site pocket, which are involved in binding of the substrate and inorganic phosphates during catalysis. This residue seems to be structurally equivalent to the conserved Arg194 in the enzyme from other sources. In the crystal both of the anion binding sites are occupied by sulphate ions. The ND atom of the catalytically important His176 is hydrogen-bonded to the main-chain carbonyl oxygen of Ser177, thus fixing the plane of the histidine imidazole ring and preventing rotation. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. A significant number of buried water molecules have been found that play an important role in the structural integrity of the molecule.