Reformable intramolecular cross-linking of the N-terminal domain of heparin cofactor II: effects on enzyme inhibition.
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 alpha-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 alpha-thrombin inhibitory activity, both in the presence and in the absence of glycosaminoglycans. Treatment with dithiothreitol and iodoacetamide restores activity towards alpha-thrombin, suggesting that release of the N terminus of HCII is an important component of the multistep interaction between the inhibitor and alpha-thrombin.
Brinkmeyer
Stephan
S
Department of Biotechnology, Faculty of Technology, University of Bielefeld, Germany.
Eckert
Ralf
R
Ragg
Hermann
H
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D002918:Chymotrypsin / Q000737:chemistry; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003488:Cyanogen Bromide / Q000737:chemistry / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D003871:Dermatan Sulfate / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D015844:Heparin Cofactor II / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D015843:Serpins / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004220:Disulfides; D004791:Enzyme Inhibitors; D006025:Glycosaminoglycans; D011994:Recombinant Proteins; D015843:Serpins; D003871:Dermatan Sulfate; D015844:Heparin Cofactor II; D006493:Heparin; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D013917:Thrombin; D003545:Cysteine; D003488:Cyanogen Bromide; D010100:Oxygen