SYNTHESIS, ACTIVITY, AND PRELIMINARY STRUCTURE OF THE 4TH EGF-LIKE DOMAIN OF THROMBOMODULIN

The fourth EGF-like domain of thrombomodulin (TM4), residues E346-F389 in the TM sequence, has been synthesized. Refolding of the synthetic product under redox conditions gave a single major product. The disulf ide bonding pattern of the folded, oxidized domain was (1-3, 2-4, 5-6) , which is the same as that found in EGF protein. TM4 was tested for T M anticoagulant activity because deletion and substitution mutagenesis experiments have shown that the fourth EGF-like domain of TM is essen tial for TM cofactor activity. TM4 showed no TM-like activity in two a ssay systems, both for inhibition of fibrin clot formation, and for co factor activity in thrombin activation of protein C. A preliminary str ucture of TM4 was determined by 2D H-1 NMR from 519 NOE-derived distan ce constraints. Distance geometry calculations yielded a single conver gent structure. The structure resembles the structure of EGF and other known EGF-like domains but has some key differences. The central two- stranded beta-sheet is conserved despite the differences in the number of amino acids in the loops. The C-terminal loop formed by the disulf ide bond between C372 and C386 in TM4 is five amino acids longer than the analogous loop between C33 and C42 of EGF protein. This loop appea rs to have a different fold in TM4 than in EGF protein. The loop forms the two outside strands of a broken, irregular tri-stranded beta-shee t, and amino acids H384-F389 lie between the two strands forming the m iddle strand of the sheet. Thus, although the C-terminus of EGF protei n forms one of the outside strands of a tri-stranded antiparallel shee t, the C-terminus of TM4 forms the inside strand of an irregular tri-s tranded parallel-antiparallel sheet. The residues D349, E357, and E374 , which were shown to be critical for cofactor activity by alanine sca nning mutagenesis, all lie in a patch near the C-terminal loop, and ar e solvent accessible. The other critical residues, Y358 and F376, are largely buried and appear to play essential structural rather than fun ctional roles.