G. Deng et al., STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE PLASMINOGEN-ACTIVATOR INHIBITOR-1 BINDING MOTIF IN THE SOMATOMEDIN-B DOMAIN OF VITRONECTIN, The Journal of biological chemistry, 271(22), 1996, pp. 12716-12723
Plasminogen activator inhibitor 1 (PAI-1) binds to the somatomedin B (
SMB) domain of vitronectin (VN), a domain present in at least seven ot
her proteins. In this study, we investigate the PAI-1 binding activity
of these SMB homologs and attempt to more specifically localize the P
AI-1 binding site within this domain. SMB(VN) and several of its homol
ogs were expressed in Escherichia coli, purified, and tested for PAI-1
binding activity in a competitive ligand binding assay. Although reco
mbinant SMB(VN) was fully active in this assay, none of the homologs b
ound to PAT-1 or competed with VN for PAI-1 binding. These inactive ho
mologs are structurally related to SMB(VN), having 33-45% sequence ide
ntity and containing all 8 cysteines at conserved positions. Thus, hom
olog-scanning experiments were conducted by exchanging progressively l
arger portions of the NH2- or COOH-terminal regions of active SMB(VN)
with the corresponding regions of the inactive homologs. These experim
ents revealed that the minimum PAI-1-binding sequence was present in t
he central region (residues 12-30) of SMB(VN). Alanine scanning mutage
nesis further demonstrated that each of the 8 cysteines as well as Gly
(12), Asp(22), Leu(24), Try(27), Tyr(28) and Asp(34) were critical for
PAI-1 binding and were required to stabilize PAI-1 activity. These re
sults indicate that the PAI-1 binding motif is localized to residues 1
2-30 of SMB(VN) and suggest that this motif is anchored in the active
conformation by disulfide bonds.