NMR STRUCTURE DETERMINATION OF TICK ANTICOAGULANT PEPTIDE (TAP)

Tick anticoagulant peptide (TAP) is a potent and selective 60-amino ac id inhibitor of the serine protease Factor Xa (fXa), the penultimate e nzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but t he binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of ser ine protease inhibition and could provide a basis for the design of no vel fXa inhibitors. Analyses of home- and heteronuclear two-dimensiona l NMR spectra (total correlation spectroscopy, nuclear Overhauser effe ct spectroscopy [NOESY], constant time heteronuclear single quantum co rrelation spectroscopy [CT-HSQC], and HSQC-NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, C-13-labeled, and N-15-labeled TAP provided nea rly complete H-1 sequence-specific resonance assignments. Secondary st ructural elements were identified by characteristic NOE patterns and D 2O amide proton-exchange experiments. A three-dimensional structure of TAP was generated from 412 NOESY-derived distance and 47 dihedral ang le constraints. The structural elements of TAP are similar in some res pects to those of the Kunitz serine protease inhibitor family, with wh ich TAP shares weak sequence homology. This structure, coupled with pr evious kinetic and biochemical information, confirms previous suggesti ons that TAP has a unique mode of binding to fXa.