A NEW STRUCTURAL CLASS OF SERINE-PROTEASE INHIBITORS REVEALED BY THE STRUCTURE OF THE HIRUSTASIN-KALLIKREIN COMPLEX

Background: Hirustasin belongs to a class of serine protease inhibitor s characterized by a well conserved pattern of cysteine residues. Unli ke the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation o f the pattern of cysteine residues and the significant sequence homolo gy suggest that these related inhibitors possess a similar three-dimen sional structure to hirustasin. Results: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 A ngstrom resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary stru ctural elements, The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar mo tifs, As a hydrophobic core is absent from hirustasin the disulfide br idges maintain the tertiary structure and present the primary binding loop to the active site of the protease, The general structural topogr aphy and disulfide connectivity of hirustasin has not previously been described. Conclusion: The crystal structure of the kallikrein-hirusta sin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, m aking it the prototype for a new class of inhibitor. The disulfide pat tern shows that the structure consists of two domains, but only the C- terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other protein s. Kallikrein recognizes hirustasin by the formation of an antiparalle l beta sheet between the protease and the inhibitor. The P1 arginine b inds in a deep negatively charged pocket of the enzyme, An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.