16 research outputs found
THE INTACT AND CLEAVED HUMAN ANTITHROMBIN-III COMPLEX AS A MODEL FOR SERPIN-PROTEINASE INTERACTIONS
THE INTACT AND CLEAVED HUMAN ANTITHROMBIN-III COMPLEX AS A MODEL FOR SERPIN-PROTEINASE INTERACTIONS
Antithrombin is a member of the serine proteinase inhibitor (serpin) family which contain a flexible reactive site loop that interacts with, and is cleaved by the target proteinase. In cleaved and latent serpins, the reactive site loop is inserted into a large central beta-sheet in the same molecule, whereas in ovalbumin, a nonfunctional serpin, the reactive site loop is completely exposed and in an alpha-heliacal conformation. however in neither conformation can the reactive site loop bind to target proteinases. here we report the structure of an intact and cleaved human antithrombin complex. The intact reactive site loop is in a novel conformation that seems well suited for interaction with proteinases such as thrombin and blood coagulation factor Xa
Synthesis and biological evaluation of a backbone-modified phytoalexin elicitor
Two suitably protected building blocks (11 and 33) for the preparation of amide-linked heptaglucoside mimetic 2, an analogue of the naturally occurring phytoalexin elicitor 1a, were readily accessible by glycal chemistry. Sequential elongation of terminal glucuronide 21 with laminaribiosyl hemiaminal 33 and anomeric amine 11 by EDC/HOBt-catalyzed condensation and two-step conversion of the C6-OTr moiety into the corresponding carboxylate function afforded homogeneous carbopeptoid 2 in high overall yield. It was found that replacement of the acetal linkages by the more rigid amide bonds destroys the phytoalexin-elicitor activity
Expression of a bioactive, single-chain choriogonadotropin in Dictyostelium discoideum
Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non-covalently associated subunits. We aimed for the expression of a single-chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single-chain hCG is indeed produced by Dictyostelium. Furthermore, this single-chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptor-binding affinity is comparable to single-chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins
X-ray structure of antistasin at 1.9 angstrom resolution and its modelled complex with blood coagulation factor Xa
The three-dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 Angstrom resolution by X-ray crystallography, The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55-residue protease inhibitor, However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two beta-strands, and does not inhibit factor Xa, The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations, Consequently, the domain shapes are different, the N-terminal domain being wedge-shaped and the C-terminal domain flat, Docking studies suggest that differences in domain shape enable the N-terminal, but not C-terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site, Furthermore, a putative exosite binding region could be defined in the N-terminal domain of antistasin, comprising residues 15-17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa, In the C-terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain