TAILIEUCHUNG - Báo cáo khoa học: Reformable intramolecular cross-linking of the N-terminal domain of heparin cofactor II

The crystal structure of a heparin cofactor II (HCII)– thrombin Michaelis complex has revealed extensive con-tacts encompassing the N-terminal domain of HCII and exosite I of the proteinase. In contrast, the location of the N-terminal extension in the uncomplexed inhibitor was unclear. Using a disulfide cross-linking strategy, we dem-onstrate that at least three different sites (positions 52, 54 and 68) within the N terminus may be tethered in a reformable manner to position 195 in the loop region between helix D and strand s2A of the HCII molecule, suggesting that the N-terminal domain may interact with the inhibitor scaffold in a permissive manner | Eur. J. Biochem. 271 4275-4283 2004 FEBS 2004 doi Reformable intramolecular cross-linking of the N-terminal domain of heparin cofactor II Effects on enzyme inhibition Stephan Brinkmeyer Ralf Eckert and Hermann Ragg Department of Biotechnology Faculty of Technology University of Bielefeld Germany The crystal structure of a heparin cofactor II HCII -thrombin Michaelis complex has revealed extensive contacts encompassing the N-terminal domain of HCII and exosite I of the proteinase. In contrast the location of the N-terminal extension in the uncomplexed inhibitor was unclear. Using a disulfide cross-linking strategy we demonstrate that at least three different sites positions 52 54 and 68 within the N terminus may be tethered in a reformable manner to position 195 in the loop region between helix D and strand s2A of the HCII molecule suggesting that the N-terminal domain may interact with the inhibitor scaffold in a permissive manner. Cross-linking of the N terminus to the HCII body does not strongly affect the inhibition of a-chymotrypsin indicating that the reactive site loop sequences of the engineered inhibitor variants required for interaction with one of the HCII target enzymes are normally accessible. In contrast intramolecular tethering of the N-terminal extension results in a drastic decrease of a-thrombin inhibitory activity both in the presence and in the absence of glycosaminoglycans. Treatment with dithiothreitol and iodoacetamide restores activity towards a-thrombin suggesting that release of the N terminus of HCII is an important component of the multistep interaction between the inhibitor and a-thrombin. Keywords a-thrombin dermatan sulfate heparin cofactor II heparin serpin s . Heparin cofactor II HCII a member of the serpin family is an efficient inhibitor of a-thrombin in the presence of a variety of poly anions including glycosaminoglycan GAGs such as heparin or dermatan sulfate 1 . In the absence of these compounds

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