Biomedical subjects

T S Peat

Publications and source records attributed to T S Peat.

13 recordsLinked to original sources

PubMed2005

Structural analysis of a set of proteins resulting from a bacterial genomics project.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

Databases, Protein↗

PubMed2001

A structural genomics approach to the study of quorum sensing: crystal structures of three LuxS orthologs.

BACKGROUND: Quorum sensing is the mechanism by which bacteria control gene expression in response to cell density. Two major quorum-sensing systems have been identified, system 1 and system 2, each with a characteristic signaling molecule (autoinducer-1, or AI-1, in the case of system 1, and AI-2 in system 2). The luxS gene is required for the AI-2 system of quorum sensing. LuxS and AI-2 have been described in both Gram-negative and Gram-positive bacterial species and have been shown to be involved in the expression of virulence genes in several pathogens. RESULTS: The structure of the LuxS protein from three different bacterial species with resolutions ranging from 1.8 A to 2.4 A has been solved using an X-ray crystallographic structural genomics approach. The structure of LuxS reported here is seen to have a new alpha-beta fold. In all structures, an equivalent homodimer is observed. A metal ion identified as zinc was seen bound to a Cys-His-His triad. Methionine was found bound to the protein near the metal and at the dimer interface. CONCLUSIONS: These structures provide support for a hypothesis that explains the in vivo action of LuxS. Specifically, acting as a homodimer, the protein binds a methionine analog, S-ribosylhomocysteine (SRH). The zinc atom is in position to cleave the ribose ring in a step along the synthesis pathway of AI-2.

Amino Acid Sequence↗

PubMed2001

Solution structure of Pyrobaculum aerophilum DsrC, an archaeal homologue of the gamma subunit of dissimilatory sulfite reductase.

The solution structure of DsrC, an archaeal homologue of the gamma subunit of dissimilatory sulfite reductase, has been determined by NMR spectroscopy. This 12.7-kDa protein from the hyperthermophilic archaeon Pyrobaculum aerophilum adopts a novel fold consisting of an orthogonal helical bundle with a beta hairpin along one side. A portion of the structure resembles the helix-turn-helix DNA-binding motif common in transcriptional regulator proteins. The protein contains two disulfide bonds but remains folded following reduction of the disulfides. DsrC proteins from organisms other than Pyrobaculum species do not contain these disulfide bonds. A conserved cysteine next to the C-terminus, which is not involved in the disulfide bonds, is located on a seven-residue C-terminal arm that is not part of the globular protein and is likely to dynamically sample more than one conformation.

Amino Acid Sequence↗

PubMed1999

Haloalkane dehalogenases: structure of a Rhodococcus enzyme.

The hydrolytic haloalkane dehalogenases are promising bioremediation and biocatalytic agents. Two general classes of dehalogenases have been reported from Xanthobacter and Rhodococcus. While these enzymes share 30% amino acid sequence identity, they have significantly different substrate specificities and halide-binding properties. We report the 1.5 A resolution crystal structure of the Rhodococcus dehalogenase at pH 5.5, pH 7.0, and pH 5.5 in the presence of NaI. The Rhodococcus and Xanthobacter enzymes have significant structural homology in the alpha/beta hydrolase core, but differ considerably in the cap domain. Consistent with its broad specificity for primary, secondary, and cyclic haloalkanes, the Rhodococcus enzyme has a substantially larger active site cavity. Significantly, the Rhodococcus dehalogenase has a different catalytic triad topology than the Xanthobacter enzyme. In the Xanthobacter dehalogenase, the third carboxylate functionality in the triad is provided by D260, which is positioned on the loop between beta7 and the penultimate helix. The carboxylate functionality in the Rhodococcus catalytic triad is donated from E141. A model of the enzyme cocrystallized with sodium iodide shows two iodide binding sites; one that defines the normal substrate and product-binding site and a second within the active site region. In the substrate and product complexes, the halogen binds to the Xanthobacter enzyme via hydrogen bonds with the N(eta)H of both W125 and W175. The Rhodococcusenzyme does not have a tryptophan analogous to W175. Instead, bound halide is stabilized with hydrogen bonds to the N(eta)H of W118 and to N(delta)H of N52. It appears that when cocrystallized with NaI the Rhodococcus enzyme has a rare stable S-I covalent bond to S(gamma) of C187.

Amino Acid Sequence↗

PubMed1999

Intermolecular cleavage by UmuD-like enzymes: identification of residues required for cleavage and substrate specificity.

The UmuD-like proteins are best characterized for their role in damage-induced SOS mutagenesis. An essential step in this process is the enzymatic self-processing of the UmuD-like proteins. This reaction is thought to occur either via an intramolecular or intermolecular self-cleavage mechanism. Here, we demonstrate that it can also occur via an heterologous intermolecular cleavage reaction. The Escherichia coli UmuD enzyme demonstrated the broadest substrate specificity, cleaving both E. coli and Salmonella typhimurium UmuD substrates in vivo. In comparison, the wild-type S. typhimurium UmuD (UmuDSt) and MucA enzymes catalyzed intermolecular self-cleavage, but did not facilitate heterologous cleavage. Heterologous cleavage by the UmuDSt enzyme was, however, observed with chimeric UmuD substrates that possess residues 30-55 of UmuDSt. We have further localized the residue predominantly responsible for UmuDSt-catalyzed heterologous cleavage to Ser50 in the substrate molecule. We hypothesize that changes at this residue affect the positioning of the cleavage site of a substrate molecule within the catalytic cleft of the UmuDSt enzyme by affecting the formation of a so-called UmuD "filament-dimer". This hypothesis is further supported by the observation that mutations known to disrupt an E. coli UmuD' filament dimer also block intermolecular UmuDEc cleavage.

Amino Acid Sequence↗

PubMed1998

Structure of translation initiation factor 5A from Pyrobaculum aerophilum at 1.75 A resolution.

BACKGROUND: Translation initiation factor 5A (IF-5A) is reported to be involved in the first step of peptide bond formation in translation, to be involved in cell-cycle regulation and to be a cofactor for the Rev and Rex transactivator proteins of human immunodeficiency virus-1 and T-cell leukemia virus I, respectively. IF-5A contains an unusual amino acid, hypusine (N-epsilon-(4-aminobutyl-2-hydroxy)lysine), that is required for its function. The first step in the post-translational modification of lysine to hypusine is catalyzed by the enzyme deoxyhypusine synthase, the structure of which has been published recently. RESULTS: IF-5A from the archebacterium Pyrobaculum aerophilum has been heterologously expressed in Escherichia coli with selenomethionine substitution. The crystal structure of IF-5A has been determined by multiwavelength anomalous diffraction and refined to 1.75 A. Unmodified P. aerophilum IF-5A is found to be a beta structure with two domains and three separate hydrophobic cores. CONCLUSIONS: The lysine (Lys42) that is post-translationally modified by deoxyhypusine synthase is found at one end of the IF-5A molecule in an turn between beta strands beta4 and beta5; this lysine residue is freely solvent accessible. The C-terminal domain is found to be homologous to the cold-shock protein CspA of E. coli, which has a well characterized RNA-binding fold, suggesting that IF-5A is involved in RNA binding.

Amino Acid Sequence↗

PubMed1998

Class-directed structure determination: foundation for a protein structure initiative.

The recent sequencing of many complete genomes, combined with the development of methods that allow rapid structure determination for many proteins, has changed the way in which protein structure determinations can be approached. One-by-one determinations of individual protein structures will soon be augmented by class-directed structure analyses in which a group of proteins is targeted and structures of representative members are determined and used to represent the entire group. Such a shift in approach would be the foundation for a broad protein structure initiative targeting classes of proteins important for biotechnology and for a fundamental understanding of protein function.

Animals↗

PubMed1998

A nondenaturing purification scheme for the DNA-binding domain of poly(ADP-ribose) polymerase, a structure-specific DNA-binding protein.

Poly(ADP-ribose) polymerase (PARP) is thought to be involved in DNA repair given its ability to recognize and bind to DNA strand breaks. During apoptosis, PARP is proteolytically cleaved into two stable fragments, the N-terminal 25-kDa DNA-binding domain (DBD) and the 85-kDa fragment containing the automodification and catalytic domains. To understand the DNA-binding properties of PARP, we expressed a recombinant hexahistidine tagged protein (His-DBD) in Escherichia coli. We modified expression to facilitate protein folding by including zinc and reducing the induction temperature. Properly folded, the DNA-binding domain of PARP binds to single- and double-stranded DNA in a structure-specific manner. To eliminate contamination with bacterial DNA that occurred during the purification process, a purification procedure was developed to produce DNA-free protein. In addition, to remove the hexahistidine tag from the recombinant protein, thrombin cleavage was carried out while the recombinant protein was bound to a DNA column. This procedure stabilized the recombinant protein and resulted in nearly 100% cleavage with no appreciable loss to unwanted proteolytic degradation. This nondenaturing purification scheme results in high-quality, native PARP-DBD for use in structural and biochemical studies.

Amino Acid Sequence↗

PubMed1998

Novel Escherichia coli umuD' mutants: structure-function insights into SOS mutagenesis.

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD', the function of UmuD' has yet to be determined. In an attempt to elucidate the role of UmuD' in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD' plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD' mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD', three were nonsense mutations that resulted in a truncated UmuD' protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD'. All 17 umuD' mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD'.

Bacterial Proteins↗

PubMed1996

The UmuD' protein filament and its potential role in damage induced mutagenesis.

BACKGROUND: Damage induced 'SOS mutagenesis' may occur transiently as part of the global SOS response to DNA damage in bacteria. A key participant in this process is the UmuD protein, which is produced in an inactive from but converted to the active form, UmuD', by a RecA-mediated self-cleavage reaction. UmuD', together with UmuC and activated RecA (RecA*), enables the DNA polymerase III holoenzyme to replicate across chemical and UV induced lesions. The efficiency of this reaction depends on several intricate protein-protein interactions. RESULTS: Recent X-ray crystallographic analysis shows that in addition to forming molecular dimers, the N- and C-terminal tails of UmuD' extend from a globular beta structure to associate and produce crystallized filaments. We have investigated this phenomenon and find that these filaments appear to relate to biological activity. Higher order oligomers are found in solution with UmuD', but not with UmuD nor with a mutant of UmuD' lacking the extended N terminus. Deletion of the N terminus of UmuD' does not affect its ability to form molecular dimers but does severely compromise its ability to interact with a RecA-DNA filament and to participate in mutagenesis. Mutations in the C terminus of UmuD' result in both gain and loss of function for mutagenesis. CONCLUSIONS: The activation of UmuD to UmuD' appears to cause a large conformational change in the protein which allows it to form oligomers in solution at physiologically relevant concentrations. Properties of these oligomers are consistent with the filament structures seen in crystals of UmuD'.

Amino Acid Sequence↗

PubMed1996

Structure of the UmuD' protein and its regulation in response to DNA damage.

For life to be sustained, mistakes in DNA repair must be tolerated when damage obscures the genetic information. In bacteria such as Escherichia coli, DNA damage elicits the well regulated 'SOS response'. For the extreme case of damage that cannot be repaired by conventional enzymes, there are proteins that allow the replication of DNA through such lesions, but with a reduction in the fidelity of replication. Essential proteins in this mutagenic process are RecA, DNA polymerase III, UmuD, UmuD' and UmuC (umu: UV mutagenesis). Regulation of this response involves a RecA-mediated self-cleavage of UmuD to produce UmuD'. To understand this system in more detail, we have determined the crystal structure of the E. coli UmuD' mutagenesis protein at 2.5 A resolution. Globular heads folded in an unusual Beta-structure associate to form molecular dimers, and extended amino-terminal tails associate to produce crystallized filaments. The structure provides insight into the mechanism of the self-cleavage reaction that UmuD-like proteins undergo as part of the global SOS response.

Amino Acid Sequence↗

PubMed1996

Production and crystallization of a selenomethionyl variant of UmuD', an Escherichia coli SOS response protein.

Crystals of both native and mutant Escherichia coli UmuD' protein were obtained using the hanging drop method. Soaking the native crystals in solutions of heavy metal ions failed to produce good isomorphous derivatives, and selenomethionine substituted wild-type protein did not crystallize under conditions that gave native crystals. Site-directed mutagenesis was used to change the penultimate residue, a methionine amino acid, to either a valine or a threonine amino acid. Crystals were subsequently obtained from these mutant proteins with and without selenomethionine incorporation. Crystals of the native, the mutant, and the selenomethionine incorporated protein were all similar, crystallizing in the P4(1)2(1)2 space group.

Amino Acid Sequence↗

PubMed1995

Characterization of crystals of the thermostable DNA polymerase I from Thermus aquaticus.

Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 A resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermostability in proteins.

Crystallization↗