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Z Dauter

Publications and source records attributed to Z Dauter.

At least 19 recordsLinked to original sources
PubMed2001

Inhibitor complexes of the Pseudomonas serine-carboxyl proteinase.

Crystal structures of the serine-carboxyl proteinase from Pseudomonas sp. 101 (PSCP), complexed with a number of inhibitors, have been solved and refined at high- to atomic-level resolution. All of these inhibitors (tyrostatin, pseudo-tyrostatin, AcIPF, AcIAF, and chymostatin, as well as previously studied iodotyrostatin and pseudo-iodotyrostatin) make covalent bonds to the active site Ser287 through their aldehyde moieties, while their side chains occupy subsites S1-S4 of the enzyme. The mode of binding of the inhibitors is almost identical for their P1 and P2 side chains, while significant differences are observed for P3 and P4 (if present). Kinetic parameters for the binding of these nanomolar inhibitors to PSCP have been established and correlated with the observed mode of binding. The preferences of this enzyme for a larger side chain in P2 as well as Tyr or Phe in P1 are explained by the size, shape, and characteristics of the S2 and S1 regions of the protein structure, respectively. Networks of hydrogen bonds involving glutamic and aspartic acids have been analyzed for the atomic-resolution structure of the native enzyme. PSCP contains a calcium-binding site that consists of Asp328, Asp348, three amide carbonyl groups, and a water molecule, in almost perfect octahedral coordination. The presence of Ca(2+) cation is necessary for the activity of the enzyme.

Binding Sites↗
PubMed2001

Anomalous signal of phosphorus used for phasing DNA oligomer: importance of data redundancy.

The crystal structure of a left-handed Z-DNA hexamer duplex d(CGCGCG)(2) has been solved based on the anomalous diffraction signal of inherent P atoms using data collected at the single wavelength of 1.54 A. The anomalous signal of about 2% of the total diffracted intensity, constant for all nucleic acids, may be generally useful for solving crystal structures of DNA and RNA oligomers. The multiplicity of intensity measurements is shown to crucially affect the data quality and the ability to solve the phase problem. The anisotropic model refined to an R factor of 8.9% at 0.95 A resolution.

Biopolymers↗
PubMed2001

Protein crystal structure solution by fast incorporation of negatively and positively charged anomalous scatterers.

The preparation of derivatives by the traditional methods of soaking is one of the most time-consuming steps in protein crystal structure solution by X-ray diffraction techniques. The 'quick cryosoaking' procedure for derivatization with halides (monovalent anions) offers the possibility of significantly speeding up this process [Dauter et al. (2000), Acta Cryst. D56, 232-237]. In the present work, an extension of this technique is proposed and the use of two different classes of compounds (monovalent and polyvalent cations) that can be successfully utilized in the quick cryosoaking procedure for the derivatization and phasing of protein crystals is described. This approach has been tested on hen egg-white lysozyme and has been successfully used to solve the structure of a novel trypsin inhibitor. The possibility of using cations in the fast cryosoaking procedure gives additional flexibility in the process of derivatization and increases the chances of success in phase determination. This method can be applied to high-throughput crystallographic projects.

Crystallography, X-Ray↗
PubMed2001

Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes.

The crystal structure of a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 (PSCP) has been solved by single-wavelength anomalous diffraction using the absorption peak of bromide anions. Structures of the uninhibited enzyme and of complexes with an inhibitor that was either covalently or noncovalently bound were refined at 1.0-1.4 A resolution. The structure of PSCP comprises a single compact domain with a diameter of approximately 55 A, consisting of a seven-stranded parallel beta-sheet flanked on both sides by a number of helices. The fold of PSCP is a superset of the subtilisin fold, and the covalently bound inhibitor is linked to the enzyme through a serine residue. Thus, the structure of PSCP defines a novel family of serine-carboxyl proteinases (defined as MEROPS S53) with a unique catalytic triad consisting of Glu 80, Asp 84 and Ser 287.

Amino Acid Sequence↗
PubMed2001

Practical experience with the use of halides for phasing macromolecular structures: a powerful tool for structural genomics.

The crystal structure of pepstatin-insensitive carboxyl proteinase (PCP) from Pseudomonas sp. 101, an enzyme with no overall sequence similarity to any other proteinases of known structure, was solved using crystals soaked in sodium bromide solution and then cryocooled. A data set collected at the bromine peak absorption wavelength was sufficient for calculation of an excellent map and the entire process of phasing and tracing the maps required almost no direct human intervention. The process of structure solution using single-wavelength data was compared with three-wavelength multiwavelength anomalous diffraction (MAD); although the latter resulted in slightly better maps, the use of this much more labor-intensive approach did not significantly improve the ability to solve the structure. The successful phasing approaches are compared with several less successful attempts utilizing other crystal forms of the enzyme and the practical aspects of the use of bromine as a heavy-atom derivative are discussed. In conclusion, the use of halides with single-wavelength diffraction data fulfills the requirements of being a first-choice method of high-throughput structure solution for the emerging field of structural genomics.

Aspartic Acid Endopeptidases↗
PubMed2000

Structure of the BH domain from graf and its implications for Rho GTPase recognition.

Cellular signaling by small G-proteins is down-regulated by GTPase-activating proteins (GAPs), which increase the rate of GTP hydrolysis. The GTPase regulator associated with focal adhesion kinase (Graf) exhibits GAP activity toward the RhoA and Cdc42 GTPases, but is only weakly active toward the closely related Rac1. We determined the crystal structure of a 231-residue fragment of Graf (GrafGAP), a domain containing the GAP activity, at 2.4-A resolution. The structure clarifies the boundaries of the functional domain and yields insight to the mechanism of substrate recognition. Modeling its interaction with substrate suggested that a favorable interaction with Glu-95 of Cdc42 (Glu-97 of RhoA) would be absent with the corresponding Ala-95 of Rac1. Indeed, GrafGAP activity is diminished approximately 40-fold toward a Cdc42 E95A mutant, whereas a approximately 10-fold increase is observed for a Rac1 A95E mutant. The GrafGAP epitope that apparently interacts with Glu-95(Glu-97) contains Asn-225, which was recently found mutated in some myeloid leukemia patients. We conclude that position 95 of the GTPase is an important determinant for GrafGAP specificity in cellular function and tumor suppression.

Amino Acid Sequence↗
PubMed2000

Crystal structure of the human acyl protein thioesterase I from a single X-ray data set to 1.5 A.

BACKGROUND: Many proteins undergo posttranslational modifications involving covalent attachment of lipid groups. Among them is palmitoylation, a dynamic, reversible process that affects trimeric G proteins and Ras and constitutes a regulatory mechanism for signal transduction pathways. Recently, an acylhydrolase previously identified as lysophospholipase has been shown to function as an acyl protein thioesterase, which catalyzes depalmitoylation of Galpha proteins as well as Ras. Its amino acid sequence suggested that the protein is evolutionarily related to neutral lipases and other thioesterases, but direct structural information was not available. RESULTS: We have solved the crystal structure of the human putative Galpha-regulatory protein acyl thioesterase (hAPT1) with a single data set collected from a crystal containing the wild-type protein. The phases were calculated to 1.8 A resolution based on anomalous scattering from Br(-) ions introduced in the cryoprotectant solution in which the crystal was soaked for 20 s. The model was refined against data extending to a resolution of 1.5 A to an R factor of 18.6%. The enzyme is a member of the ubiquitous alpha/beta hydrolase family, which includes other acylhydrolases such as the palmitoyl protein thioesterase (PPT1). CONCLUSIONS: The human APT1 is closely related to a previously described carboxylesterase from Pseudomonas fluorescens. The active site contains a catalytic triad of Ser-114, His-203, and Asp-169. Like carboxylesterase, hAPT1 appears to be dimeric, although the mutual disposition of molecules in the two dimers differs. Unlike carboxylesterase, the substrate binding pocket and the active site of hAPT1 are occluded by the dimer interface, suggesting that the enzyme must dissociate upon interaction with substrate.

Acylation↗
PubMed2000

Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.

Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.

Acarbose↗
PubMed2000

Substrate specificity in glycoside hydrolase family 10. Structural and kinetic analysis of the Streptomyces lividans xylanase 10A.

Endoxylanases are a group of enzymes that hydrolyze the beta-1, 4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 A resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 A, which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The alpha-linked intermediate with the nucleophile, Glu-236, is in a (4)C(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.

Glycoside Hydrolases↗
PubMed2000

Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD.

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0. 132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.

Amino Acid Sequence↗
PubMed2000

Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme.

Here we report the solution and refinement at 1.9 A resolution of the crystal structure of the Escherichia coli medium chain length acyl-CoA thioesterase II. This enzyme is a close homolog of the human protein that interacts with the product of the HIV-1 Nef gene, sharing 45% amino acid sequence identity with it. The structure of the E. coli thioesterase II reveals a new tertiary fold, a 'double hot dog', showing an internal repeat with a basic unit that is structurally similar to the recently described beta-hydroxydecanoyl thiol ester dehydrase. The catalytic site, inferred from the crystal structure and verified by site directed mutagenesis, involves novel chemistry and includes Asp 204, Gln 278 and Thr 228, which synergistically activate a nucleophilic water molecule.

Amino Acid Sequence↗
PubMed2000

Effects of crystal twinning on the ability to solve a macromolecular structure using multiwavelength anomalous diffraction.

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved by multiwavelength anomalous diffraction (MAD) for a selenomethionine (SeMet) derivative of the protein at 1.8 A resolution, using crystals in space group P2(1) [Yang et al. (2000), Nature Struct. Biol. 7, 230-237]. Subsequent analysis showed that the crystals of both the original protein and the SeMet derivative were pseudo-merohedrally twinned with a twinning fraction approximately 0.36, owing to the near-identity of the a and c axes. An analysis of the crystal structure solution is presented and the utility of twinned crystals for solving the structure using MAD and of different phasing strategies is discussed; the results obtained with several software packages are compared.

Capsid↗
PubMed2000

Novel approach to phasing proteins: derivatization by short cryo-soaking with halides.

A quick (less than 1 min) soak of protein crystals in a cryo-solution containing bromide or iodide anions leads to incorporation of these anomalous scatterers into the ordered solvent region around the protein molecules. These halide anions provide a convenient way of phasing through their anomalous scattering signal: bromides using multiwavelength anomalous dispersion (MAD) and bromides and/or iodides using single-wavelength anomalous dispersion (SAD) or single isomorphous replacement with anomalous scattering (SIRAS) methods. This approach has been tested successfully on four different proteins and has been used to solve the structure of a new protein of molecular weight 30 kDa.

Anions↗
PubMed1999

Atomic resolution structures of the core domain of avian sarcoma virus integrase and its D64N mutant.

Six crystal structures of the core domain of integrase (IN) from avian sarcoma virus (ASV) and its active-site derivative containing an Asp64 --> Asn substitution have been solved at atomic resolution ranging 1.02-1.42 A. The high-quality data provide new structural information about the active site of the enzyme and clarify previous inconsistencies in the description of this fragment. The very high resolution of the data and excellent quality of the refined models explain the dynamic properties of IN and the multiple conformations of its disordered residues. They also allow an accurate description of the solvent structure and help to locate other molecules bound to the enzyme. A detailed analysis of the flexible active-site region, in particular the loop formed by residues 144-154, suggests conformational changes which may be associated with substrate binding and enzymatic activity. The pH-dependent conformational changes of the active-site loop correlates with the pH vs activity profile observed for ASV IN.

Amino Acid Substitution↗
PubMed1999

Experimental observation of bonding electrons in proteins.

We demonstrate with two examples the success and potential of recent developments in x-ray protein crystallography at ultra high resolution. Our preliminary structural analyses using diffraction data collected for the two proteins crambin and savinase show meaningful deviations from the conventional independent spherical atom approximation. A noise-reduction averaging technique enables bonding details of electron distributions in proteins to be revealed experimentally for the first time. We move one step closer to imaging directly the fine details of the electronic structure on which the biological function of a protein is based.

Animals↗