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W Kabsch

Publications and source records attributed to W Kabsch.

At least 19 recordsLinked to original sources
PubMed1995

The actin fold.

X-ray structure analysis of actin and of the NH2-terminal domain of the heat-shock cognate protein Hsc70 has revealed an unexpected extensive structural similarity between these two molecules. Despite the absence of significant similarity of their amino acid sequences, both proteins share the same core architecture and a common nucleotide binding site resembling the structure of hexokinase. All three are ATPases or kinases and bind ATP in association with Mg2+ or Ca2+. The common fold consists of two alpha/beta domains, which are connected by a putative hinge with an ATP-binding site situated between the domains. Each domain contains a five-stranded beta-sheet of identical topology, which suggests that the molecules may have evolved by gene duplication. From a comparison of the three aligned structures, a fingerprint sequence of the adenine nucleotide binding pocket was derived, which predicted that members of the glycerol kinase family should also have a similar fold of their nucleotide binding domain. This was later confirmed when the X-ray structure was published. Data base search with a refined consensus sequence has retrieved other sugar kinases, as well as the prokaryotic cell cycle proteins FtsA, MreB, and StbA, and two Escherichia coli phosphatases. These proteins are predicted to possess a structure similar to actin in the common core region. As exemplified for actin, Hsc70, and glycerol kinase, the diversity of biological function is provided by the polymorphism of the loops joining the beta-strands and helices in the core region and by inserted domains that show high variability.

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↗
PubMed1984

On the use of sequence homologies to predict protein structure: identical pentapeptides can have completely different conformations.

The search for amino acid sequence homologies can be a powerful tool for predicting protein structure. Discovered sequence homologies are currently used in predicting the function of oncogene proteins. To sharpen this tool, we investigated the structural significance of short sequence homologies by searching proteins of known three-dimensional structure for subsequence identities. In 62 proteins with 10,000 residues, we found that the longest isolated homologies between unrelated proteins are five residues long. In 6 (out of 25) cases we saw surprising structural adaptability: the same five residues are part of an alpha-helix in one protein and part of a beta-strand in another protein. These examples show quantitatively that pentapeptide structure within a protein is strongly dependent on sequence context, a fact essentially ignored in most protein structure prediction methods: just considering the local sequence of five residues is not sufficient to predict correctly the local conformation (secondary structure). Cooperativity of length six or longer must be taken into account. Also, we are warned that in the growing practice of comparing a new protein sequence with a data base of known sequences, finding an identical pentapeptide sequence between two proteins is not a significant indication of structural similarity or of evolutionary kinship.

Amino Acid Sequence↗
PubMed1983

How good are predictions of protein secondary structure?

The three most widely used methods for the prediction of protein secondary structure from the amino acid sequence are tested on 62 proteins of known structure using a program package and data collection not previously available. None of these methods predicts better than 56% of the residues correctly, for a three state model (helix, sheet and loop). The algorithms of Robson et al. [J. Mol. Biol. (1978) 120, 97-120] and Lim [J. Mol. Biol. (1974) 88, 873-894] are the best of those tested. New methods, now under development, can be tested against this benchmark.

Amino Acid Sequence↗
PubMed1982

The ten helical twist angles of B-DNA.

On the assumption that the twist angles between adjacent base-pairs in the DNA molecule are additive a linear system of 40 equations was derived from experimental measurements of the total twist angles for different pieces of DNA of known sequences. This system of equations is found to be statistically consistent providing a solution for all ten possible twist angles of B-DNA by a least squares fitting procedure. Four of the calculated twist angles were not known before (tau AC, tau AG, tau CA, tau TA). The other six twist angles calculated are very close to the experimentally measured ones (tau AA, tau AT, tau CC, tau OG, tau GA, tau GC). The data used were obtained by the electrophoretic band-shift method (1-3), crystallography (4) and nuclease digestion of DNA adsorbed to mica or Ca-phosphate surface (5,6). The validity of the principle of additivity of the twist angles implies that the angle between any particular two base-pairs is a function of only these base-pairs, independent of nearest neighbours.

DNA↗