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R E Stenkamp

Publications and source records attributed to R E Stenkamp.

At least 37 records · Page 2Linked to original sources
PubMed1997

Crystal structure of a 30 kDa C-terminal fragment from the gamma chain of human fibrinogen.

BACKGROUND: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, (alphabetagamma)2, held together by 29 disulfide bonds. The globular C terminus of the gamma chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. RESULTS: The X-ray crystallographic structure of the 30 kDa globular C terminus of the gamma chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. CONCLUSIONS: The polymerization domain in the gamma chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.

Amino Acid Sequence↗
PubMed1997

Structural studies of the streptavidin binding loop.

The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.

Allosteric Regulation↗
PubMed1997

Protein superfamily members as targets for computer modeling: the carbohydrate recognition domain of a macrophage lectin.

Members of protein superfamilies display similar folds, but share only limited sequence identity, often 25% or less. Thus, it is not straightforward to apply standard homology modeling methods to construct reliable three-dimensional models of such proteins. A three-dimensional model of the carbohydrate recognition domain of the rat macrophage lectin, a member of the calcium-dependent (C-type) lectin superfamily, has been generated to illustrate how information provided by comparison of X-ray structures and sequence-structure alignments can aid in comparative modeling when primary sequence similarities are low.

Amino Acid Sequence↗
PubMed1996

Structure and function studies of factor XIIIa by x-ray crystallography.

The three-dimensional structures of several forms of the factor XIII A subunit have been determined using single crystal x-ray diffraction methods. Our crystallographic studies have provided the first detailed structural view of the factor XIII A subunit and information that is useful for understanding transglutaminase function. We have identified a conserved Cys314-His373-Asp396 catalytic triad of residues in the active site of the molecule and a number of other conserved residues that may play important roles as well. The calcium and strontium structures have revealed several conserved acidic residues (Asp438, Glu485, and Glu490) involved in ion binding. We have also been able to use our crystal structures as scaffolds to model the possible structural effects of missense mutations that have been identified in factor XIII-deficient patients.

Binding Sites↗
PubMed1995

Structural evidence that the activation peptide is not released upon thrombin cleavage of factor XIII.

The three-dimensional structure of the recombinant human factor XIII a2 dimer after cleavage by thrombin has been determined by X-ray crystallography. Factor XIII zymogen was treated with bovine alpha-thrombin in the presence of 3 mM CaCl2, and the cleaved protein was crystallized from Tris buffered at pH 6.5 using ethanol as the precipitating agent. Refinement of the molecular model of thrombin-cleaved factor XIII against diffraction data from 10.0 to 2.5 A resolution has been carried out to give a crystallographic R factor of 18.2%. The structure of thrombin-cleaved factor XIII is remarkably similar to that of the zymogen: there are no large conformational changes in the protein and the 37 residue amino terminus activation peptide remains associated with the rest of the molecule. This work shows that the activation peptide, upon thrombin cleavage, has the same conformation and occupies the same position with respect to the rest of the molecule as it does in the zymogen structure.

Animals↗
PubMed1994

Three-dimensional structure of a transglutaminase: human blood coagulation factor XIII.

Mechanical stability in many biological materials is provided by the crosslinking of large structural proteins with gamma-glutamyl-epsilon-lysyl amide bonds. The three-dimensional structure of human recombinant factor XIII (EC 2.3.2.13 zymogen; protein-glutamine:amine gamma-glutamyltransferase a chain), a transglutaminase zymogen, has been solved at 2.8-A resolution by x-ray crystallography. This structure shows that each chain of the homodimeric protein is folded into four sequential domains. A catalytic triad reminiscent of that observed in cysteine proteases has been identified in the core domain. The amino-terminal activation peptide of each subunit crosses the dimer interface and partially occludes the opening of the catalytic cavity in the second subunit, preventing substrate binding to the zymogen. A proposal for the mechanism of activation by thrombin and calcium is made that details the structural events leading to active factor XIIIa'.

Catalysis↗
PubMed1994

The corn inhibitor of blood coagulation factor XIIa. Crystallization and preliminary crystallographic analysis.

A 13.6 kDa protein from corn seeds is known to be a highly selective inhibitor of human blood coagulation Factor XIIa (or activated Hageman factor). We have crystallized this inhibitor at 23 degrees C and pH 7.5 from a solution of 30% polyethylene glycol 400, 0.2 M MgCl2, and 0.1 M Hepes. The crystals diffract to at least 2.1 A resolution. The space group is P4(2)2(1)2 with a = b = 57.15 A and c = 80.5 A. The crystals contain 51% solvent. Two heavy atom derivatives have been identified.

Cloning, Molecular↗
PubMed1994

Transglutaminase factor XIII uses proteinase-like catalytic triad to crosslink macromolecules.

The X-ray crystal structure of human transglutaminase factor XIII has revealed a cysteine proteinase-like active site involved in a crosslinking reaction and not proteolysis. This is among the first observations of similar active sites in 2 different enzyme families catalyzing a similar reaction in opposite directions. Although the size and overall protein fold of factor XIII and the cysteine proteinases are quite different, the active site and the surrounding protein structure share structural features suggesting a common evolutionary lineage. Here we present a description of the residues in the active site and the structural evidence that the catalytic mechanism of the transglutaminases is similar to the reverse mechanism of the cysteine proteinases.

Binding Sites↗
PubMed1992

Structural studies of oxygen-bridged iron compounds.

Compound (I): cis-dichloro[mu-[bis[mu-[[2,6-diacetylpyridine dioximato](2-)-O:O']]dihydroxodiphenyl-diborato](3-)]- mu- mithoxydiiron, [Fe2(C30H29B2N6-O6)Cl2(CH3O)], M(r) = 804.9, monoclinic, Cc, a = 21.228 (6), b = 8.020 (2), c = 20.865 (5) A, beta = 105.2 (1) degrees, V = 3428 (2) A3, Z = 4, Dx = 1.56 g cm-3, lambda(Mo K alpha) = 0.7107 A, mu = 10.6 cm-1, F(000) = 1648, T = 275 K, R = 0.055 for 3018 unique reflections. Compound (I) contains a pseudo-twofold axis relating the two pyridine dioxime groups bound to the Fe atoms. In addition, two O atoms from the phenylborate moieties bridge the iron atoms, as does a methoxide. The two Cl atoms are bound to the seven coordinate metals axial to the methoxide. Compound (II): cis-dichloro[mu-[bis[mu-[[2,6-diacetylpyridine dioximato](2-)-O:O']]dihydroxodiphenyldiborato](3-)]-mu- hydroxodiiron, [Fe2(C30H29B2N6-O6)Cl2(OH)].H2O.2C2H3N, M(r) = 891.0, monoclinic, P2(1)/n, a = 11.860 (2), b = 20.911 (5), c = 16.175 (3) A, beta = 92.88 (1) degrees, V = 4006 (3) A3, Z = 4, Dx = 1.48 g cm-3, lambda(Mo K alpha) = 0.7107 A, mu = 9.1 cm-1, F(000) = 1832, T = 275 K, R = 0.051 for 7034 unique reflections. Compound (II) contains a hydroxide group replacing the methoxide in compound (I). The crystals also contain two acetonitriles of solvation. The bond lengths in the complex and the hydrogen-bonding pattern in the crystals are consistent with one of the bridging borate O atoms being protonated.(ABSTRACT TRUNCATED AT 250 WORDS)

Bridged-Ring Compounds↗
PubMed1991

Structures of met and azidomet hemerythrin at 1.66 A resolution.

The crystallographic refinement of met and azidomet hemerythrin has been carried out at 1.66 A resolution in an attempt to characterize precisely the binuclear iron center in this protein. Restrained least-squares refinement has produced molecular models giving R-values of 18.9% for met (65,683 reflections from 10 A to 1.66 A) and 17.6% for azidomet hemerythrin (68,747 reflections from 10.0 A to 1.66 A). The protein structure in each derivative is very similar to that of myohemerythrin. The mu-oxo bridged iron center differs between the two forms. The complex in met hemerythrin is asymmetric with the bridging oxygen closer to one of the iron atoms while the complex in azidomet hemerythrin is symmetric. After investigations of the effects of correlation in the refinement, we believe this difference between the two complexes is associated with chemical differences and is not a refinement artefact.

Animals↗
PubMed1991

Structures of deoxy and oxy hemerythrin at 2.0 A resolution.

The crystallographic structure analyses of deoxy and oxy hemerythrin have been carried out at 2.0 A resolution to extend the low resolution views of the physiological forms of this oxygen-binding protein. Restrained least-squares refinement has produced molecular models giving R-values of 16.8% for deoxy (41,064 reflections from 10 A to 2.0 A) and 17.3% for oxy hemerythrin (40,413 reflections from 10.0 A to 2.0 A). The protein structure in each derivative is very similar to that of myohemerythrin and the various met forms of hemerythrin. The binuclear complex in each derivative retains an oxygen atom bridging the two iron atoms, but the bond lengths found in deoxy hemerythrin support the idea that, in that form, the bridge is protonated, i.e. the bridging group is a hydroxyl. Dioxygen binds to the pentaco-ordinate iron atom in deoxy hemerythrin in the conversion to oxy hemerythrin. The interatomic distances are consistent with the proposed mechanism where the proton from the bridging group is transferred to the bound dioxygen, stabilizing it in the peroxo oxidation state by forming a hydrogen bond between the peroxy group and the bridging oxygen atom.

Binding Sites↗
PubMed1990

Human recombinant factor XIII from Saccharomyces cerevisiae. Crystallization and preliminary x-ray data.

Crystals of human recombinant factor XIII from the yeast Saccharomyces cerevisiae have been grown from solutions of ammonium sulfate at pH 5.8. The crystals are orthorhombic, with space group P2(1)2(1)2 and unit cell dimensions gamma a = 101.2, b = 182.7, and c = 93.4 A. The asymmetric unit consists of one a2 dimer of molecular mass 166 kDa. A 3.5-A resolution data set for the native protein has been collected. Practical resolution limits for these crystals have not been determined, but reflections have been observed to a Bragg spacing of 2.8-A resolution.

Crystallization↗
PubMed1990

The structure of rubredoxin from Desulfovibrio desulfuricans strain 27774 at 1.5 A resolution.

The structure of a small rubredoxin from the bacterium Desulfovibrio desulfuricans has been determined and refined at 1.5 A resolution. The hairpin loop containing seven residues in other rubredoxins is missing in this 45 residue molecule, and once that fact was determined by amino acid sequencing studies, refinement progressed smoothly to an R value of 0.093 for all reflections from 5 to 1.5 A resolution. Nearly all of the water molecules in the well-ordered triclinic unit cell have been added to the crystallographic model. As in the other refined rubredoxin models, the Fe-S4 complex is slightly distorted from ideal tetrahedral coordination.

Amino Acid Sequence↗
PubMed1986

Structure of rubredoxin from the bacterium Desulfovibrio desulfuricans.

The X-ray crystallographic structure of rubredoxin from Desulfovibrio desulfuricans strain 27774 is described. This molecule is 15% smaller than previously studied rubredoxins, lacking a seven-residue loop of chain but containing a histidine and a free-sulfhydryl cysteine. Except for solvent exposure of the single invariant tryptophan, no other major difference occurs in the molecule.

Amino Acids↗
PubMed1986

Novel fucolipids of human adenocarcinoma: monoclonal antibody specific for trifucosyl Ley (III3FucV3FucVI2FucnLc6) and a possible three-dimensional epitope structure.

Immunization of mice with Ley-active trifucosylnonaosylceramide (III3FucV3FucVI2FucnLc6) isolated from human colonic adenocarcinoma (Nudelman, E., Levery, S. B., Kaizu, T., and Hakomori, S. (1986) J. Biol. Chem. 261, 11247-11253) followed by selection of hybridoma by positive reaction with this antigen and negative reaction with two other Ley antigens (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) resulted in successful isolation of the hybridoma producing IgM antibody, termed KH1, specific to Ley-active trifucosylnonaosylceramide, which does not cross-react with Ley-active hexaosyl- or octaosylceramides (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) without internal fucosyl substitution. The three-dimensional structure of the trifucosylnonaosylceramide was simulated based on previously published glycosidic torsion angles for fucosyl type 2 chain (Lex and Ley) and for GlcNAc beta 1----3Gal beta as predicted by hard sphere exo-anomeric calculations (Thøgersen, H., Lemieux, R. U., Bock, K., and Meyer, B. (1982) Can. J. Chem. 60, 44-57). The picture thus constructed showed a broad nonpolar area consisting of the hydrophobic surface of the pyranosyl ring and acetamido group of N-acetylglucosamine and three CH3 groups of L-fucose; this hydrophobic area is adjacent to a hydrophilic area. In analogy to the detailed structure of Leb or Ley involved in their interactions with antibodies and lectins (Spohr, U., Hindsgaul, O., and Lemieux, R. U. (1985) Can. J. Chem. 63, 2644-2652), such a wide hydrophobic area adjacent to a hydrophilic region could be recognized by the antibody KH1, as shown in the model illustrated in the text. Since the axis of ceramide, which is inserted in the lipid bilayer, is perpendicular to the plane of type 2 chain, the epitope recognized by the antibody KH1 is located at the external nonpolar surface of the carbohydrate chain that is overlaid on the lipid bilayer.

Adenocarcinoma↗
PubMed1985

Active site structures of deoxyhemerythrin and oxyhemerythrin.

The physiologically active forms of the nonheme-iron, oxygen-transport protein hemerythrin have been studied by x-ray crystallographic techniques. At 3.9-A resolution, a difference electron-density map between the deoxy form and met form (methemerythrin) of the protein suggests only small differences in the binuclear iron complexes. The coordination of the iron atoms appears to be the same in both the deoxy and met forms, one iron of the complexes being pentacoordinate, the other iron being hexacoordinate. The iron atoms appear to be somewhat farther apart in the deoxy form. A 2.2-A resolution study of oxyhemerythrin shows that dioxygen binds to one iron atom--the pentacoordinate one in the met form of the protein, the same binding site found for azide in azidomethemerythrin.

Animals↗