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- W2048653605 abstract "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. 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." @default.
- W2048653605 created "2016-06-24" @default.
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- W2048653605 date "1996-05-01" @default.
- W2048653605 modified "2023-10-10" @default.
- W2048653605 title "Structural and Functional Analysis of the Plasminogen Activator Inhibitor-1 Binding Motif in the Somatomedin B Domain of Vitronectin" @default.
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- W2048653605 doi "https://doi.org/10.1074/jbc.271.22.12716" @default.
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