Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin.
Plasminogen activator inhibitor 1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin (VN), a domain present in at least seven other proteins. In this study, we investigate the PAI-1 binding activity of these SMB homologs and attempt to more specifically localize the PAI-1 binding site within this domain. SMBVN and several of its homologs were expressed in Escherichia coli, purified, and tested for PAI-1 binding activity in a competitive ligand binding assay. Although recombinant SMBVN was fully active in this assay, none of the homologs bound to PAI-1 or competed with VN for PAI-1 binding. These inactive homologs are structurally related to SMBVN, having 33-45% sequence identity and containing all 8 cysteines at conserved positions. Thus, homolog-scanning experiments were conducted by exchanging progressively larger portions of the NH2- or COOH-terminal regions of active SMBVN with the corresponding regions of the inactive homologs. These experiments revealed that the minimum PAI-1-binding sequence was present in the central region (residues 12-30) of SMBVN. Alanine scanning mutagenesis further demonstrated that each of the 8 cysteines as well as Gly12, Asp22, Leu24, Try27, Tyr28, and Asp34 were critical for PAI-1 binding and were required to stabilize PAI-1 activity. These results indicate that the PAI-1 binding motif is localized to residues 12-30 of SMBVN and suggest that this motif is anchored in the active conformation by disulfide bonds.
Deng
G
G
Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Royle
G
G
Wang
S
S
Crain
K
K
Loskutoff
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D017931:DNA Primers; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013002:Somatomedins / Q000235:genetics / Q000378:metabolism*; D019096:Vitronectin / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B; D003545:Cysteine