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K S Wilson

Publications and source records attributed to K S Wilson.

At least 109 records · Page 6Linked to original sources
PubMed1997

wARP: improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models.

wARP is a procedure that substantially improves crystallographic phases (and subsequently electron-density maps) as an additional step after density-modification methods such as solvent flattening and averaging. The initial phase set is used to create a number of dummy atom models which are subjected to least-squares or maximum-likelihood refinement and iterative model updating in an automated refinement procedure (ARP). Averaging of the phase sets calculated from the refined output models and weighting of structure factors by their similarity to an average vector results in a phase set that improves and extends the initial phases substantially. An important requirement is that the native data have a maximum resolution beyond approximately 2.4 A. The wARP procedure shortens the time-consuming step of model building in crystallographic structure determination and helps to prevent the introduction of errors.

Journal Article↗
PubMed1997

The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4'-hydroxyphenyl)propionic acid, together with site-directed mutagenesis and kinetic analysis, demonstrates that arginine 381 is required for substrate specificity.

The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a substrate analog, 3-(4'-hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the side-chain NH1 4.1 A from the 4'-OH of the analog. The structure has been deduced at 2.5 A resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 A, b = 143.91 A, and c = 59.80 A. To evaluate the role of Arg 381 in TPL catalysis, we prepared mutant proteins replacing arginine with alanine (R381A), with isoleucine (R381I), and with valine (R381V). The beta-elimination activity of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination activity with L-tyrosine as substrate. However, R381A, R381I, and R381V TPL react with S-(o-nitrophenyl)-L-cysteine, beta-chloro-L-alanine, O-benzoyl-L-serine, and S-methyl-L-cysteine and exhibit k(cat) and k(cat)/Km values comparable to those of wild-type TPL. Furthermore, the Ki values for competitive inhibition by L-tryptophan and L-phenylalanine are similar for wild-type, R381A, and R381I TPL. Rapid-scanning-stopped flow spectroscopic analyses also show that wild-type and mutant proteins can bind L-tyrosine and form quinonoid complexes with similar rate constants. The binding of 3-(4'-hydroxyphenyl)propionic acid to wild-type TPL decreases at high pH values with a pKa of 8.4 and is thus dependent on an acidic group, possibly Arg404, which forms an ion pair with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff base. R381A TPL shows only a small decrease in k(cat)/Km for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pKas with an average value of about 7.8. Thus, it is possible that Arg 381 is one of the catalytic bases previously observed in the pH dependence of k(cat)/Km of TPL with L-tyrosine [Kiick, D. M., & Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.

Arginine↗
PubMed1997

Structure of bacterial luciferase beta 2 homodimer: implications for flavin binding.

The crystal structure of the beta 2 homodimer of Vibrio harveyi luciferase has been determined to 2.5 A resolution by molecular replacement. Crystals were grown serendipitously using the alpha beta form of the enzyme. The subunits of the homodimer share considerable structural homology to the beta subunit of the alpha beta luciferase heterodimer. The four C-terminal residues that are disordered in the alpha beta structure are fully resolved in our structure. Four peptide bonds have been flipped relative to their orientations in the beta subunit of the alpha beta structure. The dimer interface of the homodimer is smaller than the interface of the heterodimer in terms of buried surface area and number of hydrogen bonds and salt links. Inspection of the subunits of our structure suggests that FMNH2 cannot bind to the beta 2 enzyme at the site that has been proposed for the alpha beta enzyme. However, we do uncover a potential FMNH2 binding pocket in the dimer interface, and we model FMN into this site. This proposed flavin binding motif is consistent with several lines of biochemical and structural evidence and leads to several conclusions. First, only one FMNH2 binds per homodimer. Second, we predict that reduced FAD and riboflavin should be poor substrates for beta 2. Third, the reduced activity of beta 2 compared to alpha beta is due to solvent exposure of the isoalloxazine ring in the beta 2 active site. Finally, we raise the question of whether our proposed flavin binding site could also be the binding site for flavin in the alpha beta enzyme.

Binding Sites↗
PubMed1997

The benefits of atomic resolution.

After a long gestation, the elucidation of the crystal structures of proteins at atomic resolution is now maturing. The use of such data for both refinement and structure solution is advancing space. The necessary technology is generally available, in terms of data collection, computing hardware and software. The structures appearing in the literature mainly relate to demonstration projects on native proteins. The importance of these alone is already obvious. Biologically significant results, in terms of ligand complexes and prosthetic groups, are just starting to emerge.

Algorithms↗
PubMed1997

Evolution of immunoglobulin-like modules in chitinases: their structural flexibility and functional implications.

BACKGROUND: Chitinase A from Serratia marcescens is a glycosyl hydrolase consisting of three distinct domains. The N-terminal domain (ChiN domain, amino acids 24-137) has an immunoglobulin-like fold. This ChiN domain is structurally similar to fibronectin type III domains (FnIII domains), which exist in other chitinases, but does not share any sequence similarity with them. RESULT: Structure comparisons of the ChiN domain and FnIII domains confirm the similar fold, but fail to establish any sequence similarity. Sequence searches and comparisons between ChiN and FnIII domain sequences show a remarkable difference between the two domains in chitinases from an evolutionary point of view. A low temperature structure of chitinase A shows that the ChiN module is flexible with respect to the catalytic body of the protein. CONCLUSIONS: We postulate that the ChiN and FnIII domains evolved independently in chitinases which share otherwise homologous catalytic domains. The flexibility of the ChiN domain, together with biochemical knowledge of the function of similar domains, leads us to propose that immunoglobulin-like folds in chitinases are involved in interactions with the chitin chain during catalysis.

Bacterial Proteins↗
PubMed1997

Cyclophosphamide and fluorouracil combined with mitoxantrone versus doxorubicin for breast cancer: superiority of doxorubicin.

PATIENTS AND METHODS: We conducted a randomized, multicenter study of intravenous cyclophosphamide 500 mg/m2 plus fluorouracil 500 mg/m2 combined with either mitoxantrone (Novantrone, Lederle Cyanamid Canada Ltd, Willowdale, Ontario) 10 mg/m2 (CNF) or doxorubicin (Adriamycin, Adria Laboratories of Canada Ltd, Mississauga, Ontario) 50 mg/m2 (CAF) every 3 weeks in advanced breast cancer. RESULTS: The response rate in 249 randomized patients was 36% with CNF (44 of 121) and 48% with CAF (62 of 128) (P = .054), with complete remissions in 10 patients (8.3%) on CNF and in 13 (10.2%) on CAF. If only fully assessable patients are considered, the response rate was 48% (44 of 91) with CNF and 60% (62 of 103) with CAF (P = .098). At time of analysis, all except 10 patients (one CNF and nine CAF) had died. The median survival time with CAF was longer than with CNF (15.2 v 10.9 months; P = .003), and time to progression was also longer with CAF (5.3 v 3.2 months; P < .03). Survival differences remained significant (P = .006) if patients who failed to meet all eligibility criteria were excluded. Favorable prognostic factors for survival in a Cox regression model included good performance status (P < .0001); less than two organ systems involved by tumor (P < .0001); no involvement of lung, liver, or brain (P < .003); involvement of bone or bone marrow (P < .009), prior surgery for breast cancer (P < .006); being premenopausal (P < .03); > or = 3 years from diagnosis until randomization on this study (P < .03); and treatment with CAF (P < .03). Alopecia > or = grade 3 was reported in 55% of patients with CAF and 12% of patients with CNF (P < .001), while other > or = grade 3 toxicities did not differ significantly. Priestman-Baum quality-of-life assessment was comparable on the two study arms. CONCLUSION: In patients with advanced breast cancer, CAF was associated with longer survival than was CNF, with an increase in alopecia, but not in other toxicities.

Adult↗
PubMed1997

Crystals of cytochrome c-553 from Bacillus pasteurii show diffraction to 0.97 A resolution.

We report here the purification and characterization of a c-type cytochrome present in the soluble fraction of the gram-positive, alkaliphilic, and highly ureolytic soil bacterium Bacillus pasteurii. The cytochrome is acidic (pI = 3.3), has a molecular mass of 9.5 kDa, and appears to dimerize in 150 mM ionic strength solution. The electronic spectrum is typical of a low-spin hexa-coordinated heme iron. Crystals of the protein in the oxidized state were grown by vapor diffusion at pH 5, by using 3.2 M ammonium sulfate as precipitant. Diffraction data at ultrahigh resolution (0.97 A) and completeness (99.9%) have been collected under cryogenic conditions, by using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with cell constants a = 37.14, b = 39.42, c = 44.02 A, and one protein monomer per asymmetric unit. Attempts to solve the crystal structure by ab initio methods are in progress.

Bacillus↗
PubMed1996

Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.

The Zn(Scys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxin-encoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S bonds in Zn-rubredoxin (average length, 2.345 A) as compared with Fe-rubredoxin (average length, 2.262 A). An increase of the CA-CB-SG-M dihedral angles involving Cys-6 and Cys-39, the first cysteines of each of the Cys-Xaa-Xaa-Cys metal binding motifs, has been observed. Another consequence of the replacement of iron by zinc is that the region around residues 36-46 undergoes larger displacements than the remainder of the polypeptide chain. Despite these changes, the main features of the FeS4 site, namely a local 2-fold symmetry and the characteristic network of N-H...S hydrogen bonds, are conserved in the ZnS4 site. The Zn-substituted rubredoxin provides the first precise structure of a Zn(Scys)4 unit in a protein. The nearly identical fold of rubredoxin around iron or zinc suggests that at least in some of the sites where the metal has mainly a structural role-e.g., zinc fingers-the choice of the relevant metal may be directed by its cellular availability and mobilization processes rather than by its chemical nature.

Bacterial Proteins↗
PubMed1996

Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP.

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.

Adenosine Triphosphate↗
PubMed1996

A self-validation technique for protein structure refinement: the extended Hamilton test.

An extension is proposed for the self-validation Hamilton test [Hamilton (1965). Acta Cryst. 18, 502-510] for crystallographic refinement. The method is based on the statistical F test and evaluates the significance of the R-factor ratio between two refinement protocols. The general case of two refinements carried out with different numbers and types of non-linear restraints is examined. The restraints are considered as extra observations weighted by a coefficient expressing their effective number. There exists a restriction on the weighting coefficients between the two refinements. An empirical method to evaluate the effective number of restraints is provided. The method may allow the detection of unreasonably tight restraints. The expectation value for r.m.s. R(free), given the r.m.s. R, can be estimated. Thus, the significance of the observed drop in R(free) can be assessed. Compared to cross-validation using R(free) [Brünger (1992). Nature (London), 355, 472-474] self-validation has the advantage that it does not require omission of any experimental data. The significance of the improvement obtained by moving from isotropic to anisotropic description of thermal parameters in the refinement of a protein at 1.5 A, resolution is used as an example.

Journal Article↗
PubMed1996

X-ray structure of yeast inorganic pyrophosphatase complexed with manganese and phosphate.

The three-dimensional structure of the manganese-phosphate complex of inorganic pyrophosphatase from Saccharomyces cerevisiae has been refined to an R factor of 19.0% at 2.4-A resolution. X-ray data were collected from a single crystal using an imaging plate scanner and synchrotron radiation. There is one dimeric molecule in the asymmetric unit. The upper estimate of the root-mean-square coordinate error is 0.4 A using either the delta A plot or the superposition of the two crystallographically independent subunits. The good agreement between the coordinates of the two subunits, which were not subjected to non-crystallographic symmetry restraints, provides independent validation of the structure analysis. The active site in each subunit contains four manganese ions and two phosphates. The manganese ions are coordinated by the side chains of aspartate and glutamate residues. The phosphate groups, which were identified on the basis of their local stereochemistry, interact either directly or via water molecules with manganese ions and lysine, arginine, and tyrosine side chains. The phosphates are bridged by two of the manganese ions. The outer phosphate is exposed to solvent. The inner phosphate is surrounded by all four manganese ions. The ion-binding sites are related to the order of binding previously established from kinetic studies. A hypothesis for the transition state of the catalytic reaction is put forward.

Crystallography, X-Ray↗
PubMed1996

Specificity mechanisms in the control of transcription.

In this overview we analyze and illustrate the principles underlying some of the specificity mechanisms that control the initiation, elongation, and termination phases of transcription. Thermodynamic mechanisms dominate in the first steps of initiation, where promoters at various levels of activation can be considered to be in competition for a limiting supply of core RNA polymerase. In the later stages of initiation, as well as in elongation and termination, the regulatory mechanisms that control specificity are largely kinetic, involving rate competition between branching reaction pathways where the outcome depends on the rates (and equilibria) of reaction and interconversion of different forms of the transcription complex. Elongation complexes are very stable at most positions along the DNA template, meaning that only RNA chain elongation (and editing) can occur at these positions. However, the stability of transcription complexes decreases abruptly when termination sequences are encountered, and here the outcome can be easily switched between elongation and termination (RNA release) by minor changes in the relative rates of these competing processes. Cis effectors, defined as sites at which regulatory proteins bind to upstream activation loci on either the DNA or the nascent RNA, play important roles in the control of both initiation and of the elongation-termination decision. Examples, drawn from studies of phage lambda N-dependent antitermination and E. coli rho-dependent termination processes, illustrate the flexibility and additivity of regulatory components within control mechanisms in transcription that involve multiple determinants. The generality of such regulatory principles are stressed.

Animals↗
PubMed1996

Structure of the heme d of Penicillium vitale and Escherichia coli catalases.

A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma-spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.

Catalase↗
PubMed1996

Ribonuclease from Streptomyces aureofaciens at atomic resolution.

Crystals of ribonuclease from Streptomyces aureofaciens diffract to atomic resolution at room temperature. Using synchrotron radiation and an imaging-plate scanner, X-ray data have been recorded to 1.20 A resolution from a crystal of native enzyme and to 1.15 A from a crystal of a complex with guanosine-2'-monophosphate. Refinement with anisotropic atomic temperature factors resulted in increased accuracy of the structure. The R factors for the two structures are 10.6 and 10.9%. The estimated r.m.s. error in the coordinates is 0.05 A, less than half that obtained in the previous analysis at 1.7 A resolution. For the well ordered part of the main chain the error falls to below 0.02 A as estimated from inversion of the least-squares matrix. The two independent molecules in the asymmetric unit allowed detailed analysis of peptide planarity and some torsion angles. The high accuracy of the analysis revealed density for a partially occupied anion in the nucleotide binding site of molecule A in the native structure which was not seen at lower resolution. The anisotropic model allowed correction of the identity of the residue at position 72 from cysteine to threonine. Cys72 SG had been modelled in previous analyses with two conformations. The solvent structure was modelled by means of an automated procedure employing a set of objective criteria. The solvent structure for models refined using different programs with isotropic and anisotropic description of thermal motion is compared.

Journal Article↗
PubMed1996

How to escape from model bias with a high-resolution native data set - structure determination of the PcpA-S6 subunit III.

The structure of procarboxypeptidase A-S6 subunit III, a truncated zymogen E, has been determined by molecular replacement using as search model porcine elastase 1 which, as revealed by crystallographic analysis, contained about 20% of the amino acids in a radically different orientation. Two monoclinic crystal forms were used: the first one diffracts to 2.3 A resolution and contains one molecule per asymmetric unit; the second diffracts to 1.7 A resolution and contains two molecules per asymmetric unit. Molecular replacement and conventional X-PLOR refinement led to a model for which 20% of the chain was ill defined in both crystal forms. To remove the bias introduced by the initial model, an automated refinement procedure [Lamzin & Wilson (1993). Acta Cryst. D49, 129-147] was applied successfully to the second crystal form, which diffracts to high resolution. The resulting dramatic improvement of the electron-density map led to extensive rebuilding of some surface loops. The reliability of the modified model was confirmed by refinement of the first crystal form. For the two forms, the final R factor is 18.8% for data between 8.0 and 2.0 A resolution, and 18.4% for data between 8.0 and 1.7 A, respectively.

Journal Article↗
PubMed1996

Enzymatic ketonization of 2-hydroxymuconate: specificity and mechanism investigated by the crystal structures of two isomerases.

5-Carboxymethyl-2-hydroxymuconate isomerase (CHMI) and 4-oxalocrotonate tautomerase (4-OT) are enzymes that catalyze the isomerization of unsaturated ketones. They share a common enzyme mechanism, although they show a preference for different substrates. There is no apparent sequence homology between the enzymes. To investigate the molecular mechanism and the basis for their substrate specificity, we have determined the crystal structures of the two enzymes at high resolution. 4-OT is hexameric, with the subunits arranged with 32 symmetry. CHMI is trimeric and has extensive contacts between subunits, which include secondary structural elements. The central core of the CHMI monomer has a fold similar to a 4-OT dimer, but the secondary structural elements that form the subunit contacts around the 3-fold axis are different in the two enzymes. The region of greatest similarity between the two enzymes is a large pocket that is proposed to be the active site. The enzymes appear to operate via a "one-base" mechanism, and the possible role of residues in this pocket is discussed in view of this idea. Finally, the molecular basis for substrate specificity in the two enzymes is discussed.

Binding Sites↗
PubMed1996

Three-dimensional structure of Xenopus laevis Cu,Zn superoxide dismutase b determined by X-ray crystallography at 1.5 A resolution.

Xenopus laevis Cu,Zn superoxide dismutase (recombinant isoenzyme b) has been crystallized and the structure determined at 1.49 A resolution. The crystals belong to space group P2(1)2(1)2(1), with cell constants a = 73.33, b = 68.86, c = 59.73 A, and contain one dimeric molecule of M(r) 32 000 per asymmetric unit. The structure was solved by molecular-replacement techniques using the semisynthetic Cu,Co bovine enzyme as search model, and refined by molecular dynamics with a crystallographic pseudo-energy term. During the final steps, positional and anisotropic thermal parameters of the atoms were refined. The R factor for the 49 209 unique reflections in the 10.0-1.49 A resolution range is 0.104, for a model comprising 2023 protein atoms, two Cu(2+), two Zn(2+), and 353 water molecules. The overall temperature factor for the model, including solvent, is 20.3 A(2), while the calculated r.m.s. coordinate error for the refined model is 0.036 A. As suggested by the primary structure homology to any other known intracellular eukaryotic superoxide dismutase (> 50%), the typical structural scaffolding of flattened antiparallel eight-stranded (beta-barrel is well conserved in X. laevis Cu,Zn superoxide dismutase b, together with the coordination geometry of the metal centers in the active site. The higher thermal stability of the bb X. laevis superoxide dismutase homodimer, with respect to dimers involving the a-type isoenzyme subunit(s), can be related, on the basis of the high-resolution structure, to side-chain and solvent interactions centered on residue Tyr149, in both b-type subunits. The analysis of the overall solvent structure reveals a number of equivalent water molecule sites in the two subunits, and in homologous superoxide dismutase models. Their locations are discussed in detail and classified on the basis of their structural role.

Journal Article↗