Integrin alpha(2) subunit forms in the complex with the beta(1) subunit a c
ell surface receptor binding extracellular matrix molecules, such as collag
ens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are r
ecognized by the special "inserted" domain (I domain) in the integrin alpha
(2) subunit, Venom from a pit viper, Bothrops jararaca, has been shown to i
nhibit the interaction of platelet alpha(2)beta(1) integrin with collagen b
ecause of the action of a disintegrin/metalloproteinase named jararhagin. T
he finding that crude B. jararaca venom could prevent the binding of human
recombinant r alpha(2)I domain to type I collagen led us to study jararhagi
n further. Synthetic peptides representing hydrophilic and charged sequence
s of jararhagin, including the RSECD sequence replacing the well known RGD
motif in the disintegrin-like domain, were synthesized. Although the disint
egrin-like domain derived peptides failed to inhibit r alpha(2)I domain bin
ding to collagen, a basic peptide from the metalloproteinase domain proved
to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, wa
s shown to bind strongly to human recombinant alpha(2)I domain and to preve
nt its binding to type I and IV collagens and to laminin-1, Mutational anal
ysis indicated that a sequence of three amino acids, arginine-lysine-lysine
(RKK), is essential for r alpha(2)I domain binding, whereas the mutation o
f the other amino acids in the peptide had little if any effect on its bind
ing function. Importantly, the peptide was functional only in the cyclic co
nformation and its affinity was strictly dependent on the size of the cyste
ine-constrained loop. Furthermore, the peptide could not bind to alpha(2)I
domain in the absence of Mg2+, suggesting that the conformation of the I do
main was critical, as well. Cells could attach to the peptide only if they
expressed alpha(2)beta(1) integrin, and the attachment was inhibited by ant
i-integrin antibodies.