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Biomedical subjects

D H Ohlendorf

Publications and source records attributed to D H Ohlendorf.

At least 19 recordsLinked to original sources
PubMed1995

Superantigen binding to a T cell receptor beta chain of known three-dimensional structure.

The three-dimensional structure of an unglycosylated T cell antigen receptor (TCR) beta chain has recently been determined to 1.7 A resolution. To investigate whether this soluble beta chain (murine V beta 8.2J beta 2.1C beta 1) retains superantigen (SAG)-binding activity, we measured its affinity for various bacterial SAGs in the absence of MHC class II molecules. Dissociation constants (KDs) were determined using two independent techniques: surface plasmon resonance detection and sedimentation equilibrium. Specific binding was demonstrated to staphylococcal enterotoxins (SEs) B, C1, C2, and C3 and to streptococcal pyrogenic exotoxin A (SPEA), consistent with the known proliferative effects of these SAGs on T cells expressing V beta 8.2. In contrast, SEA, which does not stimulate V beta 8.2-bearing cells, does not bind the recombinant beta chain. Binding of the beta chain to SAGs was characterized by extremely fast dissociation rates (> 0.1 s-1), similar to those reported for certain leukocyte adhesion molecules. Whereas the beta chain bound SEC1, 2, and 3 with KDs of 0.9-2.5 microM, the corresponding value for SEB was approximately 140 microM. The much weaker binding to SEB than to SEC1, 2, or 3 was surprising, especially since SEB was found to actually be 3- to 10-fold more effective, on a molar basis, than the other toxins in stimulating the parental T cell hybridoma. We interpret these results in terms of the ability of SEC to activate T cells independently of MHC, in contrast to SEB. We have also measured SE binding to the glycosylated form of the beta chain and found that carbohydrate apparently does not contribute to recognition, even though the N-linked glycosylation sites at V beta 8.2 residues Asn24 and Asn74 are at or near the putative SAG-binding site. This result, along with the structural basis for the V beta specificity of SEs, are discussed in relation to the crystal structure of the unglycosylated beta chain.

Enterotoxins↗
PubMed1995

Crystal structure of the superantigen staphylococcal enterotoxin type A.

Staphylococcal enterotoxins are prototype superantigens characterized by their ability to bind to major histocompatibility complex (MHC) class II molecules and subsequently activate a large fraction of T-lymphocytes. The crystal structure of staphylococcal enterotoxin type A (SEA), a 27 kDa monomeric protein, was determined to 1.9 A resolution with an R-factor of 19.9% by multiple isomorphous replacement. SEA is a two domain protein composed of a beta-barrel and a beta-grasp motif demonstrating the same general structure as staphylococcal enterotoxins SEB and TSST-1. Unique for SEA, however, is a Zn2+ coordination site involved in MHC class II binding. Four amino acids including Ser1, His187, His225 and Asp227 were found to be involved in direct coordination of the metal ion. SEA is the first Zn2+ binding enterotoxin that has been structurally determined.

Binding Sites↗
PubMed1994

Structure of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa at 2.15 A resolution.

Protocatechuate 3,4-dioxygenase catalyzes the aromatic ring cleavage of 3,4-dihydroxybenzoate by incorporating both atoms of molecular oxygen to yield beta-carboxy-cis,cis-muconate. The structure of this metalloenzyme from Pseudomonas aeruginosa (now reclassified as P. putida) has been refined to an R-factor of 0.172 to 2.15 A resolution. The structure is a highly symmetric (alpha beta Fe3+)12 aggregate with a root-mean-square (r.m.s.) difference of 0.18 A among symmetry-related atoms. The tertiary structure of the two polypeptides (alpha and beta) are highly homologous (r.m.s. difference of 1.05 A over 127 C alpha atoms), suggesting that the ancestral enzyme was originally a homodimer with two active sites. Indeed, a non-functional, vestigial active site retains many of the properties of the functional active site but does not bind iron. The coordination geometry of the non-heme iron catalytic cofactor can best be described as trigonal bipyramidal with Tyr447 (147 beta) and His462 (162 beta) serving as axial ligands, and Tyr408 (108 beta), His460 (160 beta) and Wat837 serving as equitorial ligands. The active site environment has a number of basic residues that may promote binding of the acidic substrate. Within the putative active site cavity which is located between alpha and beta chains, five approximately coplanar solvent molecules suggest a position for the planar substrate Trp449 (149 beta), Ile491 (191 beta), defined by Gly14 (14 alpha) and Pro15 (15 alpha). In this position the guanidino group of Arg457 (157 beta) would be buried by the substrate, suggesting a functional role in catalysis.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary X-ray analysis of protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus.

X-ray quality single crystals of protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus were obtained by the hanging drop method. The intradiol dioxygenase crystallizes in the cubic space group I23 with unit cell dimensions a = b = c = 145.5 A. The dodecahedral crystals diffract to beyond 2.5 A resolution. The asymmetric unit contains one twelfth of the enzyme (alpha beta Fe+3)12 complex.

Acinetobacter calcoaceticus↗
PubMed1994

Preliminary crystallographic study of protocatechuate 3,4-dioxygenase from Brevibacterium fuscum.

The enzyme protocatechuate 3,4-dioxygenase from the Gram positive organism Brevibacterium fuscum crystallizes in the triclinic space group P1 with unit cell dimensions a = 96.1 A, b = 97.2 A, c = 118.1 A and alpha = 113.9 degrees, beta = 90.7 degrees, gamma = 117.8 degrees. The rod-like crystals diffract to 2.4 A resolution. Rotation function analysis suggests that there are six promoters arranged with local 32 symmetry in the asymmetric unit rather than the previously proposed pentameric complex.

Brevibacterium↗
PubMed1994

Crystallization of catechol-1,2 dioxygenase from Pseudomonas arvilla C-1.

The metalloenzyme catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 consists of three isozymes formed by combinations of two non-identical subunits; alpha alpha, alpha beta and beta beta; with molecular masses of 59,000, 63,000 and 67,000 Da, respectively. The alpha alpha isozyme crystallizes in the orthorhombic space group C222(1) with unit cell dimensions a = 62.7 A, b = 71.5 A, c = 187.1 A. The rectangular plates diffract to 2.6 A resolution. This is the first dioxygenase to be crystallized that uses catechol as a substrate. Comparison of the structure of this enzyme with protocatechuate 3,4-dioxygenase will provide basic information about the mechanisms of subunit association, substrate selectivity, and the origins of metabolic diversity in enzymes.

Catechol 1,2-Dioxygenase↗
PubMed1994

Preliminary crystallographic analysis of methane mono-oxygenase hydroxylase from Methylosinus trichosporium OB3b.

The hydroxylase component of the enzyme methane mono-oxygenase from Methylosinus trichosporium OB3b has been crystallized in the orthorhombic space group C222(1) with unit cell dimensions a = 264.5 A, b = 71.2 A, c = 139.4 A. The crystals grow as square, thick plates and diffract to beyond 2 A resolution. There is one half of the hydroxylase dimer in the asymmetric unit.

Crystallization↗
PubMed1994

Immunobiologic and biochemical properties of mutants of toxic shock syndrome toxin-1.

Toxic shock syndrome (TSS) is a multisystem illness caused mainly by Staphylococcus aureus producing TSS toxin-1 (TSST-1). A variant of TSST-1 has been isolated from ovine mastitis S. aureus. This toxin, TSST-ovine (TSST-O) is only weakly T cell mitogenic, is nonpyrogenic, does not enhance endotoxin shock, and does not cause TSS in the miniosmotic pump model. The sequence of the ovine gene (tstO) differs from the TSST-1 gene (tstH) by 14 nucleotides that change seven amino acids in the mature protein of which two are in the C-terminal half. A gene fusion containing half of both tstH and tstO was made and cloned into S. aureus. The fusion protein contained the two C-terminal amino acid differences that are in TSST-O at residues 132 and 140. The fusion protein was not T cell mitogenic and did not elicit TSS in two rabbit models. Additional experiments used mutagenesis to change the lysine residue at position 132 of TSST-O to glutamate (TSST-OK132E), as exists in TSST-1, and to change the lysine residue of the human-ovine fusion at position 132 to glutamate (TSST-11140T). Both mutants were pyrogenic, enhanced endotoxin shock, and caused TSS in the miniosmotic pump model. However, the proteins were only partially T cell mitogenic. The restoration of lethality of TSST-O and the human-ovine fusion by changing the lysine to glutamate, as exists in TSST-1, indicates that residue 132 is important in lethality. The failure to regenerate complete T cell mitogenicity of the same mutants indicates that residues 132 and 140 are important for that activity.

Amino Acid Sequence↗
PubMed1992

Resonance Raman studies of the protocatechuate 3,4-dioxygenase from Brevibacterium fuscum.

Resonance Raman studies of the protocatechuate 3,4-dioxygenase (PCD) from Brevibacterium fuscum have been carried out to take advantage of the high iron-site homogeneity of this enzyme. Native uncomplexed PCD exhibits individual resonance-enhanced nu CO and delta CH vibrations for the two tyrosinates coordinated to the active site iron center, which can be assigned to a particular residue by their excitation profiles. Of the two nu CO features observed at 1254 and 1266 cm-1, only the latter is upshifted (to 1272 cm-1) when H2O is replaced by D2O. Similarly the 1254-cm-1 feature is unaffected, while the 1266-cm-1 feature is shifted to approximately 1290 cm-1 when inhibitors such as phenolates or terephthalate bind to the active site. These observed shifts can be rationalized by the presence of hydrogen-bonding interactions with solvent in the active site cavity, which are modulated by D2O and eliminated upon inhibitor binding. Examination of the PCD crystal structure suggests that the axial tyrosine can be hydrogen bonded in the uncomplexed enzyme to water molecules present in the substrate binding pocket. The equatorial tyrosine may also be hydrogen bonded but to solvent molecules which are trapped in a pocket inaccessible to bulk solvent. These studies allow for the first time the association of particular Raman spectroscopic features, i.e., the nu CO's at 1254 and 1266 cm-1, with the equatorial and axial tyrosine residues in the PCD active site, respectively; they lay the groundwork for further Raman studies on catalytically important species to determine the roles these tyrosine residues may play in the PCD reaction cycle.

Binding Sites↗
PubMed1992

Structural studies of the retroviral proteinase from avian myeloblastosis associated virus.

The structure of the retroviral proteinase from avian myeloblastosis associated virus (MAV) has been determined and refined at 2.2 A resolution. This structure is compared with those of homologous proteinases from Rous sarcoma virus (RSV) and human immunodeficiency type 1 virus (HIV). Through comparison with the structure of a proteinase-inhibitor complex from HIV, a model of a complex between MAV proteinase and a peptide substrate has been generated. Examination of this model suggests structural basis for the diverse specifications of viral proteinases.

Amino Acid Sequence↗
PubMed1991

Crystallographic refinement of bovine pro-phospholipase A2 at 1.6 A resolution.

Bovine pro-phospholipase A2 (Mr = 14,520), trigonal, P3(1)21, a = b = 46.5, c = 102.0 A, one molecule per asymmetric unit, lambda (Cu K alpha) = 1.54 A. The model incorporating 895 protein atoms, two molecules of 2-methyl-2,4-pentanediol, and 60 solvent water molecules, was refined by restrained least squares to a residual R = 0.194 for 14,667 reflections from 5 to 1.6 A resolution.

Enzyme Precursors↗
PubMed1990

X-ray structural studies of the cytokine interleukin 1-beta.

The structure of the human cytokine, interleukin 1-beta (IL1-beta) is composed almost wholly of anti-parallel beta-sheet, organized in a three-fold repeating motif. The beta strands comprising the protein core are interconnected by 11 surface loops that form prominent features on the surface of the molecule. Comparisons of the amino acid sequences of different species of IL1-beta and the related cytokine IL1-alpha show that the majority of conserved residues form the structural core of the molecule, and that most variability occurs in the termini and surface loops that presumably bind the IL1 receptor. These results suggest that there may be some degree of flexibility in interactions made between IL1 and its cell surface receptor.

Amino Acid Sequence↗
PubMed1989

Structural origins of high-affinity biotin binding to streptavidin.

The high affinity of the noncovalent interaction between biotin and streptavidin forms the basis for many diagnostic assays that require the formation of an irreversible and specific linkage between biological macromolecules. Comparison of the refined crystal structures of apo and a streptavidin:biotin complex shows that the high affinity results from several factors. These factors include the formation of multiple hydrogen bonds and van der Waals interactions between biotin and the protein, together with the ordering of surface polypeptide loops that bury the biotin in the protein interior. Structural alterations at the biotin binding site produce quaternary changes in the streptavidin tetramer. These changes apparently propagate through cooperative deformations in the twisted beta sheets that link tetramer subunits.

Bacterial Proteins↗
PubMed1988

Structure and assembly of protocatechuate 3,4-dioxygenase.

Dioxygenases catalyse the cleavage of molecular oxygen with subsequent incorporation of both oxygen atoms into organic substrates. Some of the best-studied dioxygenases have been isolated from bacteria where they catalyse the critical ring-opening step in the biodegradation of aromatic compounds. These bacterial enzymes generally contain nonheme ferric iron as the sole cofactor. Protocatechuate 3,4-dioxygenase (3,4-PCD) was one of the first such enzymes recognized and catalyses the intradiol cleavage of protocatechuic acid by oxygen to produce beta-carboxy-cis,cis-muconic acid. Previous studies have shown that the 3,4-PCD found in Pseudomonas aeruginosa is an oligomer with a relative molecular mass (Mr) of 587,000 (587K) containing 12 copies each of alpha (22.3K) and beta (26.6K) subunits. The X-ray structure determination of 3,4-PCD reveals the catalytic iron environment required for oxygenolytic cleavage of aromatic rings and also provides a novel holoenzyme assembly with cubic 23(T) symmetry and first examples of mixed beta-barrel domains.

Amino Acid Sequence↗
PubMed1987

Crystallographic data for Streptomyces avidinii streptavidin.

Crystallization conditions are reported for Streptomyces avidinii streptavidin with and without bound biotin. X-ray examination of the free and bound crystal forms shows the streptavidin-biotin complex crystals to be most suitable for high resolution structure analysis. A complete x-ray data set to 2.6 A resolution was collected for the streptavidin-biotin crystals using a two-dimensional area detector. Reduction and analysis of the x-ray diffraction pattern show that the complex crystallizes in the tetragonal space group I4(1)22 (a = b = 98.4 A, c = 125.8 A), with half of the streptavidin tetramer in the crystallographic asymmetric unit.

Bacterial Proteins↗
PubMed1987

Determination of the quaternary structure of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

A 2.5 A resolution data set has been collected for crystals of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa. Analysis of the data using the rotation function shows that the alpha 2 beta 2 tetramers associate to form a particle with cubic 23 (T) point group symmetry. Prior to this analysis it was believed that eight tetramers associated to form the holoenzyme. The symmetry of the crystalline holoenzyme also addresses questions concerning its iron content and substrate stoichiometry.

Crystallization↗
PubMed1986

Different interactions used by Cro repressor in specific and nonspecific DNA binding.

The mode of interaction of Cro repressor with specific and nonspecific sites on DNA was explored by chemical modification and protection of lysine and tyrosine residues. Cro has 8 lysines. In the presence of DNA, lysines 32 and 56 are fully protected and lysines 21, 62, and 63 are partially protected from alkylation. However, the terminal amino group and lysines 8, 18, and 39 are not protected. Location of the protected and unprotected lysines on the three-dimensional Cro structure defines a DNA-binding region. The results provide direct experimental support for a mode of interaction between Cro and DNA, in which Cro buries its 2-fold related alpha-helices in consecutive DNA major grooves (Anderson, W. F., Ohlendorf, D. H., Takeda, Y., and Matthews, B. W. (1981) Nature 290, 754-758; Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., and Matthews, B. W. (1982) Nature 298, 718-723). In the model, the carboxyl-terminal part of Cro was tentatively presumed to interact with the DNA minor groove. Protection of lysines 62 and 63 confirms the involvement of the carboxyl terminus in DNA binding. Although nonspecific and specific DNA protect the same lysine residues, there are differences in the nature of the interaction of Cro with nonspecific and specific DNA. Cro-nonspecific DNA interaction is salt-sensitive, suggesting that the interaction is predominantly electrostatic. On the other hand, Cro-specific DNA interaction is salt-resistant, suggesting that the interaction may include nonelectrostatic components (hydrogen bonds and hydrophobic interactions) as well. Protection experiments of tyrosine residues (against iodination) suggest that the conformation of Cro repressor changes in two stages: first, when Cro binds at nonspecific sites, and, second, when Cro binds to specific sites on DNA.

Amino Acid Sequence↗