Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin
Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin
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DOI：
10.1074/jbc.271.22.12716
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发表时间：
1996-05-31
影响因子：
4.8
通讯作者：
Loskutoff, DJ
中科院分区：
文献类型：
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作者：
Deng, G;Royle, G;Loskutoff, DJ
纤溶酶原激活物抑制剂1（派-1）与玻连蛋白（VN）的生长调节蛋白B（SMB）结构域结合，所述结构域存在于至少七种其它蛋白质中。在这项研究中，我们调查派-1的结合活性，这些SMB同源物，并试图更具体地定位派-1结合位点在这个域。SMB（VN）和它的几个同系物在大肠杆菌中表达，纯化，并在竞争性配体结合试验中测试派-1结合活性。尽管重组SMB（VN）在该测定中是完全活性的，但是没有同源物结合PAT-1或与VN竞争派-1结合。这些无活性同源物在结构上与SMB（VN）相关，具有33-45%的序列同一性并且在保守位置处含有所有8个半胱氨酸。因此，同源物扫描实验通过将活性SMB（VN）的NH 2-或COOH-末端区域的逐渐更大的部分与非活性同源物的相应区域交换来进行。这些实验表明，最小派-1结合序列存在于SMB（VN）的中心区域（残基12-30）。丙氨酸扫描诱变进一步证明8个半胱氨酸中的每一个以及Gly（12）、Asp（22）、Leu（24）、Try（27）、Tyr（28）和Asp（34）对于派-1结合是关键的，并且是稳定派-1活性所需的。这些结果表明派-1结合基序定位于SMB（VN）的残基12-30，并表明该基序通过二硫键锚定在活性构象中。
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. SMB(VN) 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 SMB(VN) was fully active in this assay, none of the homologs bound to PAT-1 or competed with VN for PAI-1 binding. These inactive homologs are structurally related to SMB(VN), 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 SMB(VN) 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 SMB(VN). Alanine scanning mutagenesis 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 results indicate that the PAI-1 binding motif is localized to residues 12-30 of SMB(VN) and suggest that this motif is anchored in the active conformation by disulfide bonds.