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W Kabsch

Publications and source records attributed to W Kabsch.

At least 37 records · Page 2Linked to original sources
PubMed1994

Crystallization and preliminary X-ray structure analysis of thermally unstable p21(H-ras) guanosine complexes.

p21 is a small guanine nucleotide binding protein that is involved in intracellular signal transduction. Biochemical data suggest that the presence of the beta-phosphate is essential for strong binding of guanine nucleotides to the protein. Guanosine or GMP bind six orders of magnitude more weakly to p21 than GDP or GTP. Moreover, the thermal stability of the protein is dramatically reduced when bound to GMP or guanosine. We have crystallized C-terminally truncated forms of p21(H-ras), with guanosine or GMP bound, in the space groups P4(3)2(1)2, P2(1)2(1)2 and P2(1). The crystals diffract in the range 2.8-2.2 A. Details of the crystallization procedures, the characterization of the crystals and preliminary results of structure determination are described. An unexpected electron-density peak was found close to the position of the beta-phosphate in the phosphate-binding loop.

Journal Article↗
PubMed1994

Intron positions in actin genes seem unrelated to the secondary structure of the protein.

A catalogue of intron positions along the coding sequence was assembled from the large number of actin genes known for different eukaryotes. 36 positions in the amino acid sequence were compared with the known three-dimensional structure of actin. At least 20 but not more than 23 intron positions are at the start or end of a secondary structural element (beta-strand, alpha-helix or 3/10 helix) while eight positions interrupt such an element. Statistical analysis shows that due to the large number of end positions the boundaries of secondary structural elements are not correlated with the intron positions. In addition, the observed intron pattern seems compatible with the null hypothesis, i.e. intron positions are randomly distributed along the actin sequence.

Actins↗
PubMed1994

The structure of Trypanosoma cruzi trypanothione reductase in the oxidized and NADPH reduced state.

The three-dimensional structure of trypanothione reductase (TR) (EC 1.6.4.8) from Trypanosoma cruzi has been solved at 0.33 nm resolution by molecular replacement using the structure of C. fasciculata TR as a starting model. Elucidation of the T. cruzi TR structure represents the first step in the rational design of a drug against Chagas' disease. The structure of T. cruzi TR is compared with those of C. fasciculata TR as well as human and E. coli glutathione reductase (GR). In the FAD-binding domain, TR has two insertions, each about 10 residues long, which do not occur in GR. The first one is a rigid loop stabilizing the position of helix 91-117 which is responsible for the wider active site of TR as compared to GR. The second insertion does not occur where it is predicted by sequence alignment; rather the residues extend three strands of the 4-stranded beta-sheet by one or two residues each. This increases the number of hydrogen bonds within the sheet structure. The structure of the NADPH.TR complex has been solved at 0.33 nm resolution. The nicotinamide ring is sandwiched between the flavin ring and the side chain of Phe-198 which undergoes the same conformational change upon coenzyme binding as Tyr-197 in GR. In addition to Arg-222 and Arg-228, which are conserved in TR and GR, Tyr-221--the last residue of the second beta-sheet strand of the beta alpha beta dinucleotide binding fold--is in hydrogen bonding distance to the 2' phosphate group of NADPH.

Amino Acid Sequence↗
PubMed1993

Crystallization and preliminary crystallographic analysis of trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease.

Trypanothione reductase from Trypanosoma cruzi is the most promising target molecule for the rational design of a specific drug against Chagas' disease. The recombinant protein was purified in a single chromatographic step and crystallized. Two crystal forms suitable for X-ray diffraction analysis were obtained. Tetragonal crystals (a = b = 87.4 A, c = 152.3 A) were grown from 30% polyethylene glycol (average M(r) = 8,000) in the presence of 0.2% beta-n-octylglucoside (space group either P4(2) with one dimer or P4(2)22 with one monomer in the asymmetric unit). Monoclinic crystals (space group P2, a = 136.3 A, b = 91.1 A, c = 126.0 A, beta = 94 degrees) were grown from 1.2 M sodium citrate in the presence of 2% octanoyl-N-methyl-glucamide. They contain two dimers of the enzyme in the asymmetric unit; both crystal forms diffract to 3 A resolution.

Animals↗
PubMed1993

A comparison of the atomic model of F-actin with cryo-electron micrographs of actin and decorated actin.

We compare the atomic model calculated from the crystal structure and the X-ray fiber diagram of orientated F-actin1) with the 3-D reconstructions produced from cryo-electron microscopy of actin2). Out to 30A resolution the two structures are essentially identical. Furthermore, by combining the atomic model with the reconstruction of S1-decorated actin filaments2) one can establish the nature of the actin binding site for myosin in the rigor complex. Each myosin head binds to two actin molecules on two distinct sites. Some of the actin residues involved in each of these binding sites can be identified. Furthermore, the atomic model of actin may be combined with the reconstruction of the S1 decorated thin filament to establish the tropomyosin binding site in the rigor complex. This result is compared with the model of tropomyosin-actin derived from an analysis of the X-ray fibre diagram of a reconstituted thin filament and are shown to be very similar.

Actins↗
PubMed1991

Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607.

Several hundred million tons of toxic mercurials are dispersed in the biosphere. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase and mercuric ion reductase (MerA). The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases, catalyses the reaction NADPH + Hg(II)----NADP+ + H+ + Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), p1258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn501 and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon. These domains can be proteolytically cleaved off without changing the catalytic efficiency. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.

Bacillus↗
PubMed1991

Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein.

Although there is very little sequence identity between the two proteins, the structures of rabbit skeletal muscle actin (375-amino acid residues) and the 44-kDa ATPase fragment of the bovine 70-kDa heat shock cognate protein (HSC70; 386 residues) are very similar. The alpha-carbon positions of 241 pairs of amino acid residues that are structurally equivalent within the two proteins can be superimposed with a root-mean-square difference in distance of 2.3 A; of these, 39 residues are identical, and 56 are conservative substitutions. In addition, the conformations of ADP are very similar in both proteins. A local sequence "fingerprint," which may be diagnostic of the adenine nucleotide beta-phosphate-binding pocket, has been derived. The fingerprint identifies members of the glycerol kinase family as candidates likely to have a similar structure in their nucleotide-binding domains. The structural differences between the two molecules mainly occur in loop regions of actin known to be involved in interactions with other monomers in the actin filament or in the binding of myosin; the corresponding regions in heat shock proteins may have functions that are as yet undetermined. Placing the Ca2+ ATP of actin on the ATPase fragment structure suggests Asp-206 (corresponding to His-161 of actin) as a candidate proton acceptor for the ATPase reaction.

Actins↗
PubMed1991

Three-dimensional structure of p21 in the active conformation and analysis of an oncogenic mutant.

The three-dimensional structure of the active guanosine triphosphate (GTP)-analogue-containing complex of the H-ras-encoded p21 has been determined. It was necessary to correct the topology of p21 as published earlier. The structure analysis shows all of the interactions between protein and GTP and how the important cofactor Mg2+ is bound. From the oncogenic mutants of p21 crystallized, a Gly12 to Arg mutation has been analyzed in detail. It shows that the overall structure of the mutant is not perturbed and that the side chain of Arg12 is coming close to the gamma-phosphate for an interaction.

Allosteric Regulation↗
PubMed1990

Atomic structure of the actin:DNase I complex.

The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease I both in the ATP and ADP forms have been determined by X-ray analysis at an effective resolution of 2.8 A and 3A, respectively. The two structures are very similar. The actin molecule consists of two domains which can be further subdivided into two subdomains. ADP or ATP is located in the cleft between the domains with a calcium ion bound to the beta- or beta- and gamma-phosphates, respectively. The motif of a five-stranded beta sheet consisting of a beta meander and a right handed beta alpha beta unit appears in each domain suggesting that gene duplication might have occurred. These sheets have the same topology as that found in hexokinase.

Actins↗
PubMed1990

Atomic model of the actin filament.

The F-actin filament has been constructed from the atomic structure of the actin monomer to fit the observed X-ray fibre diagram from oriented gels of F-actin. A unique orientation of the monomer with respect to the actin helix has been found. The main interactions are along the two-start helix with a contribution from a loop extending across the filament axis provided by the molecule in the adjacent strand. There are also contacts along the left-handed genetic helix.

Actins↗
PubMed1990

Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules.

The X-ray structures of the guanine nucleotide binding domains (amino acids 1-166) of five mutants of the H-ras oncogene product p21 were determined. The mutations described are Gly-12----Arg, Gly-12----Val, Gln-61----His, Gln-61----Leu, which are all oncogenic, and the effector region mutant Asp-38----Glu. The resolutions of the crystal structures range from 2.0 to 2.6 A. Cellular and mutant p21 proteins are almost identical, and the only significant differences are seen in loop L4 and in the vicinity of the gamma-phosphate. For the Gly-12 mutants the larger side chains interfere with GTP binding and/or hydrolysis. Gln-61 in cellular p21 adopts a conformation where it is able to catalyze GTP hydrolysis. This conformation has not been found for the mutants of Gln-61. Furthermore, Leu-61 cannot activate the nucleophilic water because of the chemical nature of its side chain. The D38E mutation preserves its ability to bind GAP.

Adenosine Triphosphate↗
PubMed1990

Time-resolved X-ray crystallographic study of the conformational change in Ha-Ras p21 protein on GTP hydrolysis.

Crystals of Ha-Ras p21 with caged GTP at the active site have been used to investigate the conformational changes of p21 on GTP hydrolysis. The structure of the short-lived p21.GTP complex was determined by Laue diffraction methods. After GTP hydrolysis, substantial structural changes occur in the parts of the molecule implicated in the interaction with GTPase-activating protein. The trigger for this process seems to be a change in coordination of the active-site Mg2+ ion as a result of loss of the gamma-phosphate of GTP.

Binding Sites↗
PubMed1990

Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis.

The crystal structure of the H-ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R-factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61-65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma-phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in-line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.

Amino Acid Sequence↗
PubMed1989

Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation.

The crystal structure of the guanine-nucleotide-binding domain of p21 (amino acids 1-166) complexed to the guanosine triphosphate analogue guanosine-5'-(beta, gamma-imido)triphosphate (GppNp) has been determined at a resolution of 2.6 A. The topological order of secondary structure elements is the same as that of the guanine-nucleotide-binding domain of bacterial elongation factor EF-Tu. Many interactions between nucleotide and protein have been identified. The effects of point mutations and the conservation of amino-acid sequence in the guanine-nucleotide-binding proteins are discussed.

Binding Sites↗
PubMed1985

Crystallization of cytoplasmic actin in complex with deoxyribonuclease I.

Crystals of cytoplasmic (porcine liver) actin in complex with deoxyribonuclease I (DNAase I) were prepared for structural determination by X-ray-diffraction analysis. The crystallization of porcine liver actin-DNAase I complex is preceded by a brief treatment with immobilized trypsin, whereby a C-terminal tri- or di-peptide including cysteine-374 is removed from the actin without any noticeable degradation of both proteins as judged by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis. Analysis of the crystals obtained does not reveal any differences in the three-dimensional structure of porcine liver actin from its skeletal compartment at up to 0.6 nm resolution. However, in contrast with crystalline skeletal-muscle actin-DNAase I complex, heavy-atom substitution of crystals of porcine liver actin-DNAase I complex could not be achieved with methyl mercuriacetate. Evidence is presented that, in porcine liver actin, the N-terminal cysteine residue is not located at position no. 10, as in skeletal- and smooth-muscle actin, but most probably at position no. 17. Thus, because this site is covered by DNAase I, the cysteine becomes inaccessible to titration with 5,5'-dithiobis-(2-nitrobenzoic acid) after complex-formation with DNAase I.

Actins↗
PubMed1985

Three-dimensional structure of the complex of actin and DNase I at 4.5 A resolution.

The shape of an actin subunit has been derived from an improved 6 A map of the complex of rabbit skeletal muscle actin and bovine pancreatic DNase I obtained by X-ray crystallographic methods. The three-dimensional structure of DNase I determined independently at 2.5 A resolution was compared with the DNase I electron density in the actin:DNase map. The two structures are very similar at 6 A resolution thus leading to an unambiguous identification of actin as well as DNase I electron density. Furthermore the correct hand of the actin structure is determined from the DNase I atomic structure. The resolution of the actin structure was extended to 4.5 A by using a single heavy-atom derivative and the knowledge of the atomic coordinates of DNase I. The dimensions of an actin subunit are 67 A X 40 A X 37 A. It consists of a small and a large domain, the small domain containing the N terminus. Actin is an alpha,beta-protein with a beta-pleated sheet in each domain. These sheets are surrounded by several alpha-helices, comprising at least 40% of the structure. The phosphate peak of the adenine nucleotide is located between the two domains. The complex of actin and DNase I as found in solution (i.e., the actin:DNase I contacts which do not depend on crystal packing) was deduced from a comparison of monoclinic with orthorhombic crystals. Residues 44-46, 51, 52, 60-62 of DNase I are close to a loop region in the small domain of actin. At a distance of approximately 15 A there is a second contact in the large domain in which Glu13 of DNase I is involved. A possible binding region for myosin is discussed.

Actins↗
PubMed1984

Three-dimensional structure of bovine pancreatic DNase I at 2.5 A resolution.

The three-dimensional structure of bovine pancreatic deoxyribonuclease I (DNase I) has been determined at 2.5 A resolution by X-ray diffraction from single crystals. An atomic model was fitted into the electron density using a graphics display system. DNase I is an alpha, beta-protein with two 6-stranded beta-pleated sheets packed against each other forming the core of a 'sandwich'-type structure. The two predominantly anti-parallel beta-sheets are flanked by three longer alpha-helices and extensive loop regions. The carbohydrate side chain attached to Asn 18 is protruding by approximately 15 A from the otherwise compact molecule of approximate dimensions 45 A X 40 A. The binding site of CA2+-deoxythymidine-3',5'-biphosphate (Ca-pdTp) has been determined by difference Fourier techniques confirming biochemical results that the active centre is close to His 131. Ca-pdTp binds at the surface of the enzyme between the two beta-pleated sheets and seems to interact with several charged amino acid side chains. Active site geometry and folding pattern of DNase I are quite different from staphylococcal nuclease, the only other Ca2+-dependent deoxyribonuclease whose structure is known at high resolution. The electron density map indicates that two Ca2+ ions are bound to the enzyme under crystallization conditions.

Animals↗