Solution structure of the smallest cofactor-active fragment of thrombomodulin.
A glycosylated fragment of thrombomodulin containing two epidermal growth factor-like domains (TMEGF45) was analyzed by NMR. The 4th-domains structure of this two-domain fragment is similar to that of the individual domain previously determined. The 5th-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 5th-domain is consistent with low heteronuclear NOEs. In the individual 5th-domain, Met 388 was disordered, and key thrombin binding residues formed a hydrophobic core. By contrast, in TMEGF45, Met 388 is in the 5th-domain core, positioned by Phe 376 from the 4th-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 4th-domain, the three interdomain linker residues, and nearly all of the 5th-domain are perturbed. Thus, TMEGF45 binds thrombin by an induced fit mechanism involving a flexible 5th-domain.
Wood
M J
MJ
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, California 92093-0359, USA.
Sampoli Benitez
B A
BA
Komives
E A
EA
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004220:Disulfides / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000737:chemistry; D006031:Glycosylation; D008715:Methionine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009038:Motion; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D018583:Pliability; D017434:Protein Structure, Tertiary; D012996:Solutions; D013917:Thrombin / Q000378:metabolism; D018180:Thrombomodulin / Q000737:chemistry* / Q000378:metabolism*
D004220:Disulfides; D010446:Peptide Fragments; D012996:Solutions; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D008715:Methionine; D013917:Thrombin; D000117:Acetylglucosamine