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R L Walter

Publications and source records attributed to R L Walter.

13 recordsLinked to original sources
PubMed2000

Crystal structure of protein isoaspartyl methyltransferase: a catalyst for protein repair.

BACKGROUND: Formation of isoaspartyl residues is one of several processes that damage proteins as they age. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) is a conserved and nearly ubiquitous enzyme that catalyzes the repair of proteins damaged by isoaspartyl formation. RESULTS: We have determined the first structure of a PIMT from crystals of the T. maritima enzyme complexed to S-adenosyl-L-homocysteine (AdoHcy) and refined it to 1.8 A resolution. Although PIMT forms one structural unit, the protein can be divided functionally into three subdomains. The central subdomain closely resembles other S-adenosyl-L-methionine-dependent methyltransferases but bears a striking alteration of topological connectivity, which is not shared by any other member of this family. Rather than arranged as a mixed beta sheet with topology 6 upward arrow7 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow, the central sheet of PIMT is reorganized to 7 upward arrow6 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow. AdoHcy is largely buried between the N-terminal and central subdomains by a conserved and largely hydrophobic loop on one rim of the binding cleft, and a conserved Ser/Thr-rich beta strand on the other. The Ser/Thr-rich strand may provide hydrogen bonds for specific interactions with isoaspartyl substrates. The side chain of Ile-206, a conserved residue, crosses the cleft, restricting access to the donor methyl group to a deep well, the putative isoaspartyl methyl acceptor site. CONCLUSIONS: The structure of PIMT reveals a unique modification of the methyltransferase fold along with a site for specific recognition of isoaspartyl substrates. The sequence conservation among PIMTs suggests that the current structure should prove a reliable model for understanding the repair of isoaspartyl damage in all organisms.

Amino Acid Sequence↗
PubMed2000

Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex.

BACKGROUND: Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-gammaRalpha) and a species-specific accessory factor (AF-1 or IFN-gammaRbeta). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. RESULTS: The crystal structure of the complex of human IFN-gamma with the soluble, glycosylated extracellular part of IFN-gammaRalpha has been determined at 2.9 A resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-gamma dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor-ligand complex thereby forming a 3:1 complex. CONCLUSIONS: The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-gamma cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of beta-sheet packing interactions between pairs of IFN-gammaRalpha receptors in these crystals suggests a possible model for receptor oligomerization of Ralpha and the structurally homologous Rbeta receptors in the fully active IFN-gamma signaling complex.

Binding Sites↗
PubMed1997

Purine nucleoside phosphorylase. 2. Catalytic mechanism.

X-ray crystallography, molecular modeling, and site-directed mutagenesis were used to delineate the catalytic mechanism of purine nucleoside phosphorylase (PNP). PNP catalyzes the reversible phosphorolysis of purine nucleosides to the corresponding purine base and ribose 1-phosphate using a substrate-assisted catalytic mechanism. The proposed transition state (TS) features an oxocarbenium ion that is stabilized by the cosubstrate phosphate dianion which itself functions as part of a catalytic triad (Glu89-His86-PO4=). Participation of phosphate in the TS accounts for the poor hydrolytic activity of PNP and is likely to be the mechanistic feature that differentiates phosphorylases from glycosidases. The proposed PNP TS also entails a hydrogen bond between N7 and a highly conserved Asn. Hydrogen bond donation to N7 in the TS stabilizes the negative charge that accumulates on the purine ring during glycosidic bond cleavage. Kinetic studies using N7-modified analogs provided additional support for the hydrogen bond. Crystallographic studies of 13 human PNP-ligand complexes indicated that PNP uses a ligand-induced conformational change to position Asn243 and other key residues in the active site for catalysis. These studies also indicated that purine nucleosides bind to PNP with a nonstandard glycosidic torsion angle (+anticlinal) and an uncommon sugar pucker (C4'-endo). Single point energy calculations predicted the binding conformation to enhance phosphorolysis through ligand strain. Structural data also suggested that purine binding precedes ribose 1-phosphate binding in the synthetic direction whereas the order of substrate binding was less clear for phosphorolysis. Conservation of the catalytically important residues across nucleoside phosphorylases with specificity for 6-oxopurine nucleosides provided further support for the proposed catalytic mechanism.

Amino Acid Sequence↗
PubMed1996

Multiple wavelength anomalous diffraction (MAD) crystal structure of rusticyanin: a highly oxidizing cupredoxin with extreme acid stability.

The X-ray crystal structure of the oxidized form of the extremely stable and highly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans has been determined by the method of multiwavelength anomalous diffraction (MAD) and refined to 1.9 A resolution. Like other cupredoxins, rusticyanin is a copper-containing metalloprotein, which is composed of a core beta-sandwich fold. In rusticyanin the beta-sandwich is composed of a six- and a seven-stranded beta-sheet. Also like other cupredoxins, the copper ion is coordinated by a cluster of four conserved residues (His85, Cys138, His143, Met148) arranged in a distorted tetrahedron. Rusticyanin has a redox potential of 680 mV, roughly twice that of any other cupredoxin, and it is optimally active at pH values < or = 2. By comparison with other cupredoxins, the three-dimensional structure of rusticyanin reveals several possible sources of the chemical differences, including more ordered secondary structure and more intersheet connectivity than other cupredoxins. The acid stability and redox potential of rusticyanin may also be enhanced over other cupredoxins by a more extensive internal hydrogen bonding network and by more extensive hydrophobic interactions surrounding the copper binding site. Finally, reduction in the number of charged residues surrounding the active site may also make a major contribution to acid stability. We propose that the resulting rigid copper binding site, which is constrained by the surrounding hydrophobic environment, structurally and electronically favours Cu(I). We propose that the two extreme chemical properties of rusticyanin are interrelated; the same unique structural features that enhance acid stability also lead to elevated redox potential.

Azurin↗
PubMed1995

High-resolution macromolecular structure determination using CCD detectors and synchrotron radiation.

BACKGROUND: Synchrotron radiation sources have made impressive contributions to macromolecular crystallography. The delay in development of appropriate X-ray detectors has, however, been a significant limitation to their efficient use. New technologies, based on charge-coupled devices (CCDs), provide capabilities for faster, more accurate, automated data collection. RESULTS: A CCD-based X-ray detector has been developed for use in macromolecular crystallography and has been in operation for about one and a half years at the Cornell High Energy Synchrotron Source. It has been used for a variety of crystallographic projects, including a number of high-resolution structural studies. The statistical quality of the data, the detector's ease and efficiency of use, and the growing number of structural results illustrate the practical utility of this new detector system. CONCLUSIONS: The new detector has enhanced capabilities for measuring diffraction patterns from crystals of macromolecules, especially at high resolution, when the X-ray intensities are weak. The survey of results described here ranges from virus crystallography to weakly diffracting small-molecule structure determination and demonstrates the potential of CCD detectors when combined with synchrotron radiation sources.

Amino Acid Sequence↗
PubMed1992

Crystal structure of recombinant human interleukin-4.

The crystal structure of recombinant human interleukin-4 (rhuIL-4) was initially determined at 3.5-A resolution by multiple isomorphous replacement techniques and subsequently refined to a resolution of 2.35 A by simulated annealing. The final crystallographic R-factor, based on all data in the range 6.0-2.35 A (7470 reflections), is 0.232. Bond lengths and bond angles in the molecule have root mean square deviations from ideal values of 0.016 A and 2.4 degrees, respectively. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhuIL-4 is a four alpha-helix bundle, which composes approximately 58% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within these connections is a two-stranded antiparallel beta-sheet. Both the tertiary and secondary structures of rhuIL-4 are similar to those of human granulocyte-macrophage colony-stimulating factor. Critical regions for receptor binding are proposed.

Amino Acid Sequence↗
PubMed1975

Proton-induced x-ray emission analysis--a promising technique for studying the metal content of plants and soils.

The ease of employing proton-induced X-ray emission analysis (PIXEA) to studies relating metal content of soils to metal uptake in plants was aptly demonstrated in an investigation concerning the effect of automotive pollution on the abundance of about 16 elements accumulated in ribwort plantain and its surrounding soil. Elemental concentrations were shown to be dependent on the age of the plant leaves, as well as the distance from the roadside.

Metals↗
PubMed1975

Levels of toxic metals in marine organisms collected from Southern California coastal waters.

Emission of toxic trace metals into southern California coastal waters has resulted in the extensive accumulation of the elements within marine sediments. The current study was undertaken to evaluate concentrations of trace metals in bottom-dwelling marine fauna collected from two sampling areas. Analyses carried out on muscle samples of the dover sole (Microstomus pacificus) and the crab (Cancer anthonyi) by proton-induced x-ray emission analysis showed considerable concentrations of arsenic and selenium. Samples of gonads, digestive gland, and muscle from the crab Mursia gaudichaudii analyzed by atomic absorption spectroscopy showed elemental concentrations in muscle similar to the crab Cancer anthonyi and much higher metal levels in gonad and digestive gland. These findings suggest the need for further studies concerning the relationship between emission of metals into the marine environment and their abundance in marine fauna.

Animals↗