Molecular cloning, functional expression, and molecular modeling of bothrostatin, a new highly active disintegrin from Bothrops jararaca venom.
Disintegrins are among the most potent antagonists of several integrins. A cDNA encoding a novel disintegrin, bothrostatin, was cloned from a Bothrops jararaca cDNA library. The precursor of bothrostatin contains a pro, a metalloproteinase, and an RGD-disintegrin domain. The disintegrin domain expressed in Escherichia coli showed high inhibitory activity on collagen-induced platelet aggregation (IC(50) of 12nM), and thus it can be used as a useful tool for studies of integrin-ligand interaction. Furthermore, we used the comparative modeling approach to obtain a model of the 3D structure of bothrostatin. Our results suggest that bothrostatin adopts a globular, closed structure in solution. The RGD motif is exposed to the solution by the loop formed by residues 45-59 and is very close to the C-terminal domain forming a finger-like structure. The proximity of the RGD loop and the C-terminal residues, which is maintained by the Cys47-Cys66 bond, suggests that the C-terminal residues are involved in the ability of bothrostatin to interact with its ligands.
Fernandez
Jorge H
JH
Laboratorio Especial de Toxinologia Aplicada and CAT/CEPID, Instituto Butantan, CEP 05503-900, Sao Paulo, Brazil.
Silva
Carlos A
CA
Assakura
Marina T
MT
Camargo
Antonio C M
AC
Serrano
Solange M T
SM
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects; D017837:Bothrops / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D003198:Computer Simulation; D003435:Crotalid Venoms / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry*; D019483:Disintegrins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D045726:Metalloproteases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship
D003435:Crotalid Venoms; D019483:Disintegrins; D045726:Metalloproteases; C500152:bothrostatin, Bothrops jararaca