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.