STRUCTURE OF THE 5TH EGF-LIKE DOMAIN OF THROMBOMODULIN - AN EGF-LIKE DOMAIN WITH A NOVEL DISULFIDE-BONDING PATTERN

The structure of the fifth EGF-like domain (residues Q387 to E426) of thrombomodulin (TMEGF5) has been determined by two-dimensional NMR. TM EGF5 binds to thrombin with a K-i of 1.9 mu M and has been shown to ha ve a novel disulfide bonding pattern in a fully active fragment of TM. In EGF, the disulfide bonding pattern is (1-3,2-4,5-6), while TMEGF5 has an uncrossed (1-2,3-4,5-6) pattern. The structure of tl-Lis novel domain, determined from 483 NOE-derived distance restraints, appears t o have diverged from the common EGF-like structure. Superposition of t he 14 lowest-energy structures of TMEGF5 gives an overall r.m.s.d. of 1.09 Angstrom for the backbone atoms. The central two-stranded beta-sh eet common to all EGF-like domains is not present in TMEGF5. The A loo p, residues C390 to C395, is twisted away from interacting with the B loop, residues C399 to C407, as in EGF, and is close to the C loop, re sidues C409 to C421. This twist causes the N and C termini to be close r together in TMEGF5 than in EGF. Most of the residues that are import ant for activity lie on one face of the molecule, which is likely to b e the thrombin-binding surface of the domain. The structure of the C l oop within the domain, which is a beta-hairpin similar to EGF, is simi lar to the structure of a synthetic version of the loop bound to throm bin as determined by transferred NOE experiments. Despite the similari ty in the structures of the loops, the residues immediately following C421 are in different positions in the two structures suggesting that these ''tail'' residues may change conformation upon thrombin binding. (C) 1997 Academic Press Limited.