Solution structure of the smallest cofactor-active fragment of thrombomodulin

A glycosylated fragment of thrombomodulin containing two epidermal growth f actor-like domains (TMEGF45) was analyzed by NMR, The 4(th)-domains structu re of this two-domain fragment is similar to that of the individual domain previously determined. The 5(th)-domain, which has uncrossed disulfide bond s, is not as well determined in the two-domain fragment than the individual domain previously solved. The flexibility of the 5(th)-domain is consisten t 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 P he 376 from the 4(th)-domain, As a result, key thrombin-binding residues th at were in the core of the individual domain are expelled. Upon thrombin bi nding, chemical shifts of two residues in the 4(th)-domain, the three inter domain linker residues, and nearly all of the 5(th)-domain are perturbed. T hus, TMEGF45 binds thrombin by an induced fit mechanism involving a flexibl e 5(th)-domain.