Solution structure of the smallest cofactor-active fragment of thrombomodulin

Abstract

A glycosylated fragment of thrombomodulin containing two epidermal growth factor-like domains (TMEGF45) was analyzed by NMR. The 4^th-domains structure of this two-domain fragment is similar to that of the individual domain previously determined. The 5^th-domain, which has uncrossed disulfide bonds, is not as well determined in the two-domain fragment than the individual domain previously solved. The flexibility of the 5^th-domain is consistent with low heteronuclear NOEs. In the individual 5^th-domain, Met 388 was disordered, and key thrombin-binding residues formed a hydrophobic core. By contrast, in TMEGF45, Met 388 is in the 5^th-domain core, positioned by Phe 376 from the 4^th-domain. As a result, key thrombin-binding residues that were in the core of the individual domain are expelled. Upon thrombin binding, chemical shifts of two residues in the 4^th-domain, the three interdomain linker residues, and nearly all of the 5^th-domain are perturbed. Thus, TMEGF45 binds thrombin by an induced fit mechanism involving a flexible 5^th-domain.