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K Harlos

Publications and source records attributed to K Harlos.

At least 37 records · Page 2Linked to original sources
PubMed1996

2.0 A resolution structure of a ternary complex of pig muscle phosphoglycerate kinase containing 3-phospho-D-glycerate and the nucleotide Mn adenylylimidodiphosphate.

The crystal structure of a ternary complex of pig muscle phosphoglycerate kinase (PGK) containing 3-phosphoglycerate (3-PG) and manganese adenylylimidodiphosphate (Mn AMP-PNP) has been determined and refined at 2.0 A resolution. The complex differs from the true substrate ternary complex only in the presence of an imido- rather than an oxylink between beta- and gamma-phosphates of the bound nucleotide. The 3-PG is bound in a similar manner to that observed in binary complexes. The nucleotide is bound in a similar manner to Mg ADP except that the metal ion is coordinated by all three alpha-, beta-, and gamma-phosphates, but not by the protein. The gamma-phosphate, which is transferred in the reaction, is not bound by the protein. One further characteristic of the ternary complex is that Arg-38 moves to a position where its guanidinium group makes a triple interaction with the N-terminal domain, the C-terminal domain, and the 1-carboxyl group of the bound 3-PG. Although a hinge-bending conformation change is seen in the ternary complex, it is no larger than that observed in the 3-PG binary complex. To reduce that distance between two bound substrates to a value consistent with the direct in-line transfer known to occur in PGK, we modeled the closure of a pronounced cleft in the protein structure situated between the bound substrates. This closure suggested a mechanism of catalysis that involves the "capture" of the gamma-phosphate by Arg-38 and the N-terminus of helix-14, which has a conserved Gly-Gly-Gly phosphate binding motif. We propose that nucleophilic attack by the 1-carboxyl group of the 3-PG on the gamma-phosphorus follows the capture of the gamma-phosphate, leading to a pentacoordinate transition state that may be stabilized by hydrogen bonds donated by the NH groups in the N-terminus of helix 14 and the guanidinium group of Arg-38. During the course of the reaction the metal ion is proposed to migrate to a position coordinating the alpha- and beta-phosphates and the carboxyl group of Asp-374. The mechanism is consistent with the structural information from binary and ternary substrate complexes and much solution data, and gives a major catalytic role to Arg-38, as indicated by site-directed mutagenesis.

Adenylyl Imidodiphosphate↗
PubMed1996

Bound water structure and polymorphic amino acids act together to allow the binding of different peptides to MHC class I HLA-B53.

The structure of the human MHC class I molecule HLA-B53 complexed to two nonameric peptide epitopes (from the malaria parasite P. falciparum and the HIV2 gag protein) has been determined by X-ray crystallography at 2.3 angstrom resolution. The structures reveal the architecture of a Pro-specific B pocket common to many HLA-B alleles. Relative to other alleles, the B53 peptide-binding groove is widened by a significant (up to 1.25 angstrom) shift in the position of the alpha 1 helix. Within this groove, bound water molecules, acting in concert with the side chains of polymorphic residues, provide the functional malleability of the MHC, which enables the high affinity/low specificity binding of multiple peptide epitopes.

Amino Acid Sequence↗
PubMed1995

Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes.

Penicillin antibiotics are all produced from fermentation-derived penicillins because their chemical synthesis is not commercially viable. The key step in penicillin biosynthesis, in which both the beta-lactam and thiazolidine rings of the nucleus are created, is mediated by isopenicillin N synthase (IPNS), which binds ferrous iron and uses dioxygen as a cosubstrate. In a unique enzymatic step, with no chemical precedent, IPNS catalyses the transfer of four hydrogen atoms from its tripeptide substrate to dioxygen forming, in a single reaction, the complete bicyclic nucleus of the penicillins. We now report the structure of IPNS complexed with manganese, which reveals the active site is unusually buried within a 'jelly-roll' motif and lined by hydrophobic residues, and suggest how this structure permits the process of penicillin formation. Sequence analyses indicate IPNS, 1-aminocyclopropane-1-carboxylic acid oxidase and many of the 2-oxo-acid-dependent oxygenases contain a conserved jelly-roll motif, forming a new structural family of enzymes.

Acremonium↗
PubMed1995

The refined structure of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens at 1.94 A.

BACKGROUND: Methanol dehydrogenase (MDH) is a bacterial periplasmic quinoprotein; it has pyrrolo-quinoline quinone (PQQ) as its prosthetic group, requires Ca2+ for activity and uses cytochrome cL as its electron acceptor. Low-resolution structures of MDH have already been determined. RESULTS: The structure of the alpha 2 beta 2 tetramer of MDH from Methylobacterium extorquens has now been determined at 1.94 A with an R-factor of 19.85%. CONCLUSIONS: The alpha-subunit of MDH has an eight-fold radial symmetry, with its eight beta-sheets stabilized by a novel tryptophan docking motif. The PQQ in the active site is held in place by a coplanar tryptophan and by a novel disulphide ring formed between adjacent cysteines which are bonded by an unusual non-planar trans peptide bond. One of the carbonyl oxygens of PQQ is bonded to the Ca2+, probably facilitating attack on the substrate, and the other carbonyl oxygen is out of the plane of the ring, confirming the presence of the predicted free-radical semiquinone form of the prosthetic group.

Alcohol Oxidoreductases↗
PubMed1995

Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 A resolution.

The cell-surface glycoprotein vascular cell adhesion molecule-1 (VCAM-1; ref. 1) mediates intercellular adhesion by specific binding to the integrin very-late antigen-4 (VLA-4, alpha 4 beta 1; ref. 3). VCAM-1, with the intercellular adhesion molecules ICAM-1, ICAM-2, ICAM-3 and the mucosal vascular addressin MAd-CAM-1, forms an integrin-binding subgroup of the immunoglobulin superfamily. In addition to their clinical relevance in inflammation, these molecules act as cellular receptors for viral and parasitic agents. The predominant form of VCAM-1 in vivo has an amino-terminal extracellular region comprising seven immunoglobulin-like domains. Functional studies have identified a conserved integrin-binding motif in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the immunoglobulin superfamily subgroup. We report here the crystal structure of a VLA-4-binding fragment composed of the first two domains of VCAM-1. The integrin-binding motif (Q38IDSPL) is highly exposed and forms the N-terminal region of the loop between beta-strands C and D of domain 1. This motif exhibits a distinctive conformation which we predict will be common to all the integrin-binding IgSF molecules. These, and additional data, map VLA-4 binding to the face of the CFG beta-sheet, the surface previously identified as the site for intercellular adhesive interactions between members of the immunoglobulin superfamily.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary X-ray diffraction characterisation of both a native and selenomethionyl VLA-4 binding fragment of VCAM-1.

Soluble fragments of the extracellular region of vascular cell adhesion molecule 1 (VCAM-1) expressed in Escherichia coli retain functional adhesive activity. An integrin (VLA-4) binding fragment consisting of the N-terminal two immunoglobulin-like domains (VCAM-d1,2) has been crystallized. The crystals belong to space group P2(1)2(1)2(1) with cell dimensions of a = 52.7 A, b = 66.5 A, c = 113.2 A and contain two molecules in the crystallographic asymmetric unit. A batch of protein produced in the standard E. coli strain (HW1110), but grown in the presence of selenomethionine enriched media, showed 85% incorporation of selenium in place of sulphur at methionine residues. The selenomethionyl VCAM-d1,2 was crystallized by microseeding techniques initially using the native crystals for nucleation. Both native and selenomethionyl crystals diffract X-rays to a minimum Bragg spacing of 1.8 A.

Cell Adhesion Molecules↗
PubMed1994

Crystal structure of the extracellular region of human tissue factor.

Tissue factor is a cell-surface glycoprotein receptor which initiates the blood coagulation cascade after vessel injury by interacting with blood clotting factor VII/VIIa and which is implicated in various pathological processes. When bound to tissue factor, factor VII is readily converted to the active protease factor VIIa by trace amounts of factors Xa, IXa or VIIa. Human tissue factor consists of 263 residues, the first 219 of which comprise the extracellular region. We have determined the crystal structure of the extracellular region at a resolution of 2.2 A. Tissue factor consists of two immunoglobulin-like domains associated through an extensive, novel, interdomain interface region. The binding site for factor VII lies at the interface region and involves residues from domain 1 and an extended loop (binding 'finger') of domain 2. This is the first reported structure of a representative of the class 2 cytokine receptor family, which also includes interferon-alpha, interferon-gamma (refs 2, 3) and interleukin-10 (ref. 4) receptors.

Amino Acid Sequence↗
PubMed1994

Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A resolution.

BACKGROUND: The T-lymphocyte antigen CD2 is an adhesion molecule implicated in immune responses in vivo. The extracellular regions of the human and rat homologues of CD2 share only 45% sequence identity and bind different protein ligands. Comparison of the human and rat soluble CD2 (sCD2) structures should provide insights into the structural basis of cell surface recognition. RESULTS: We therefore determined the crystal structure of a form of human sCD2 with single N-acetylglucosamine residues at each glycosylation site to 2.5 A resolution with an R-factor of 19.3%. It is composed of two immunoglobulin superfamily domains similar to those of rat sCD2, but the relative orientation of the domains in the two homologues differs by up to 20 degrees. An interaction involving the flat, highly charged, ligand binding GFCC'C" faces of crystallographically related human sCD2 molecules duplicates, in a different lattice, that observed in the rat sCD2 crystals. CONCLUSIONS: Intramolecular flexibility appears to be a conserved feature of CD2. The head-to-head interaction between molecules represents a general model for interactions between adhesion molecules of this structural class. Ligand specificity may be influenced by the distribution of charged residues on the binding face.

Amino Acid Sequence↗
PubMed1994

Structural basis of superantigen action inferred from crystal structure of toxic-shock syndrome toxin-1.

Superantigens stimulate T cells bearing particular T-cell receptor V beta sequences, so they are extremely potent polyclonal T-cell mitogens. T-cell activation is preceded by binding of superantigens to class II major histocompatibility complex (MHC) molecules. To further the structural characterization of these interactions, the crystal structure of a toxin associated with toxic-shock syndrome, TSST-1, which is a microbial superantigen, has been determined at 2.5 A resolution. The N- and C-terminal domains of the structure both contain regions involved in MHC class II association; the C-terminal domain is also implicated in binding the T-cell receptor. Despite low sequence conservation, the TSST-1 topology is similar to the structure reported for the superantigen staphylococcal enterotoxin B4. But TSST-1 lacks several of the structural features highlighted as central to superantigen activity in the staphylococcal enterotoxin B and we therefore reappraise the structural basis of superantigen action.

Amino Acid Sequence↗
PubMed1994

X-ray structure of PQQ-dependent methanol dehydrogenase.

The three-dimensional structure of the PQQ-dependent quinoprotein, methanol dehydrogenase from Methylobacterium extorquens AM1, has been determined at 3A resolution. The a2b2 tetrameric enzyme has a large a-chain of almost spherical form with a chain fold in which eight 4-stranded antiparallel b-sheets segments are arranged radially around a pseudo 8-fold molecular symmetry axis. The much smaller b-chain is surprisingly not globular, but has an extended conformation running across the surface of the alpha-subunit. The PQQ prosthetic group is buried within the large a-subunit located on the pseudo 8-fold molecular symmetry axis. It is surrounded by protein side-chains but not covalently bound. Associated with the PQQ are two unexpected features: a vicinal disulphide bridge formed between Cys103 and Cys104, and a calcium ion bound between the protein and the PQQ. Vicinal disulphide bridges forming highly distorted structures containing a cis peptide bond, have been proposed to be present in one or two enzymes but have not previously been available for detailed structural investigation. Activity studies have indicated that the ability of the enzyme to transfer electrons derived from the reduction of the alcohols to the specific cytochrome CL receptor is lost when the vicinal disulphide bridge is reduced. The roles of the calcium ions and the b-chain in the enzyme's activity remain to be determined.

Alcohol Oxidoreductases↗
PubMed1994

The active site of methanol dehydrogenase contains a disulphide bridge between adjacent cysteine residues.

Adjacent cysteine residues can only form disulphide bridges in a distorted structure containing a cis-peptide link. Such bridges are extremely uncommon, identified so far in the acetyl choline receptor alone where the structure of the bridge is undetermined. Here we present the first molecular description of a disulphide bridge of this type in the quinoprotein methanol dehydrogenase from Methylobacterium extorquens. We show that this structure occurs in close proximity to the pyrrolo-quinoline quinone prosthetic group and a calcium ion in the active site of the enzyme. This unusual disulphide bridge appears to play a role in the electron transfer reaction mediated by methanol dehydrogenase.

Alcohol Oxidoreductases↗
PubMed1993

Crystallization and preliminary X-ray analysis of human tissue factor extracellular domain.

The extracellular domain (residues 1 to 220) of human tissue factor has been cloned and expressed in Escherichia coli and purified to isoelectric homogeneity. Single crystals suitable for X-ray analysis have been obtained by vapour diffusion. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 45.2 A, c = 231.5 A, contain one molecule per asymmetric unit and diffract to 2.6 A resolution. Native and derivative data sets have been collected to 3.6 and 3.9 A, respectively.

Crystallography, X-Ray↗
PubMed1993

X-ray crystallographic structure of a papain-leupeptin complex.

The three-dimensional structure of the papain-leupeptin complex has been determined by X-ray crystallography to a resolution of 2.1 A (overall R-factor = 19.8%). The structure indicates that: (i) leupeptin contacts the S subsites of the papain active site and not the S' subsites; (ii) the 'carbonyl' carbon atom of the inhibitor is covalently bound by the Cys-25 sulphur atom of papain and is tetrahedrally coordinated; (iii) the 'carbonyl' oxygen atom of the inhibitor faces the oxyanion hole and makes hydrogen bond contacts with Gln-19 and Cys-25.

Amino Acid Sequence↗
PubMed1992

Crystal structure at 2.8 A resolution of a soluble form of the cell adhesion molecule CD2.

The crystal structure of a soluble form of the T lymphocyte antigen CD2 provides the first complete view of the extracellular region of a cell adhesion molecule. The topology of the molecule, which comprises two immunoglobulin-like domains, is the same as that of the first two domains of CD4 but the relative domain orientation is altered by a fairly flexible linker region. The putative ligand-binding beta-sheet forms a flat surface towards the top of the molecule. Crystal contacts between these surfaces suggest a plausible model for the adhesive interaction.

Amino Acid Sequence↗
PubMed1992

Crystallization and preliminary crystallographic investigation of methanol dehydrogenase from Methylobacterium extorquens AM1.

Single crystals of methanol dehydrogenase (MDH) from Methylobacterium extorquens AM1 have been grown by the vapour diffusion method. These crystals diffract to beyond 2 A resolution and are suitable for X-ray crystallography. They belong to the orthorhombic space group P2(1)2(1)2(1) and have the following unit cell parameters: a = 66.79 A, b = 108.9 A, c = 188.9 A. One asymmetric unit contains an alpha 2 beta 2 tetramer of MDH and the location of the non-crystallographic 2-fold symmetry axis of this tetramer is defined by the paired positions of the binding sites of heavy atoms in four MDH-derivatives.

Alcohol Oxidoreductases↗
PubMed1992

Crystal structure of the binary complex of pig muscle phosphoglycerate kinase and its substrate 3-phospho-D-glycerate.

Pig muscle phosphoglycerate kinase has been crystallized from polyethyleneglycol in the presence of its substrate 3-phospho-D-glycerate (3-PG) and the structure has been determined at 2.0 A resolution. The structure was solved using the known structure of the substrate-free horse muscle enzyme and has been refined to a crystallographic R-factor of 21.5%. 3-Phospho-D-glycerate is bound to the N-domain of the enzyme through a network of hydrogen bonds to a cluster of basic amino acid residues and by electrostatic interactions between the negatively charged phosphate and these basic protein side chains. This binding site is in good agreement with earlier proposals [Banks et al., Nature (London) 279:773-777, 1979]. The phosphate oxygen atoms are hydrogen bonded to His-62, Arg-65, Arg-122, and Arg-170. The 2-hydroxyl group, which defines the D-isomer of 3PG, is hydrogen bonded to Asp-23 and Asn-25. The carboxyl group of 3-PG points away from the N-domain towards the C-domain and is hydrogen bonded via a water molecule to main chain nitrogen atoms of helix-14. The present structure of the 3-PG-bound pig muscle enzyme is compared with the structure of the substrate-free horse enzyme. Major changes include an ordering of helix-13 and a domain movement, which brings the N-domain closer to the ATP-binding C-domain. This domain movement consists of a 7.7 degree rotation, which is less than previously estimated for the ternary complex. Local changes close to the 3-PG binding site include an ordering of Arg-65 and a shift of helix-5.

Adenosine Triphosphate↗
PubMed1991

Crystallization and preliminary crystallographic study of cathepsin D inhibitor from potatoes.

Single crystals of the glycosylated inhibitor of cathepsin D and trypsin isolated from potato tubers were obtained using the hanging drop vapor diffusion method and ammonium nitrate as precipitant. The crystals exhibit strong F222 pseudo symmetry but belong to the orthorhombic space group C222 or C222(1), with cell parameters a = 73.8 A, b = 119.9 A and c = 133.2 A with two molecules per asymmetric unit. The crystals diffract to a resolution of 2.4 A.

Cathepsin D↗