Schlott B, Gührs K H, Hartmann M, Röcker A, Collen D
Institute for Molecular Biotechnology, Jena, 07745 Germany.
J Biol Chem. 1998 Aug 28;273(35):22346-50. doi: 10.1074/jbc.273.35.22346.
Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys10-Lys11 peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH2-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH2-terminal region Lys11-Gly-Asp-Asp-Ala-Ser16-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis. Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH2-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH2-terminal amino acid substituted with Lys-generated derivatives from which the NH2-terminal initiation Met was no longer removed, yielding inactive (</= 10%) Sak42DDeltaN11(M),G12K, active (>50%) Sak42DDeltaN14(M), A15K and Sak42DDeltaN15(M),S16K, and inactive Sak42DDeltaN16(M),Y17K. Lys variants without NH2-terminal Met, generated from fusion proteins in which a His6 tag and a factor Xa recognition sequence were linked to the NH2 terminus of the Sak variants, were indistinguishable from their NH2-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DDeltaN11(M),G12K could be restored by additional substitution of both Asp13 and Asp14 with Asn, yielding active Sak42DDeltaN11(M),G12K, D13N, D14N, whereas substitution in Sak42DDeltaN16(M),Y17K of Phe18 and Glu19 with Asn yielded inactive Sak42DDeltaN16(M),Y17K,F18N,E19N. These data, in combination with the recent finding that the 20 NH2-terminal amino acids of Sak lack secondary structure, suggest that the NH2-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH2-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.
葡萄球菌激酶（Sak）与纤溶酶原形成1:1化学计量的无活性复合物，该复合物需要将纤溶酶原转化为纤溶酶以及水解Sak的Lys10 - Lys11肽键才能成为有效的纤溶酶原激活剂（施洛特，B.，古尔斯，K.-H.，哈特曼，M.，罗克，A.，和科伦，D.（1997年）《生物化学杂志》272，6067 - 6072）。Sak水解后带正电荷的NH2末端氨基酸的暴露是纤溶酶原激活潜力的主要决定因素，但它本身既不是必需的也不是充分的。在此，通过缺失和取代诱变研究了加工后的Sak的NH2末端区域Lys11 - Gly - Asp - Asp - Ala - Ser16 - Tyr - Phe - Glu中对于纤溶酶原激活潜力所必需的结构基序。在大肠杆菌中表达缺失11、14、15或16个NH2末端氨基酸的变体产生了正确加工但无活性的分子。表达其同源物，其中NH2末端氨基酸被Lys取代，产生的衍生物中NH2末端起始的Met不再被去除，产生无活性（≤10%）的Sak42DDeltaN11(M),G12K、活性（>50%）的Sak42DDeltaN14(M),A15K和Sak42DDeltaN15(M),S16K以及无活性的Sak42DDeltaN16(M),Y17K。从融合蛋白产生的无NH2末端Met的Lys变体，其中His6标签和因子Xa识别序列与Sak变体的NH2末端相连，与它们含NH2末端Met的对应物没有区别。通过生物特异性相互作用分析测量，所有研究的变体对纤溶酶原都具有完整的亲和力。Sak42DDeltaN11(M),G12K的活性可以通过将Asp13和Asp14都额外替换为Asn来恢复，产生活性的Sak42DDeltaN11(M),G12K,D13N,D14N，而在Sak42DDeltaN16(M),Y17K中用Asn取代Phe18和Glu19产生无活性的Sak42DDeltaN16(M),Y17K,F18N,E19N。这些数据，结合最近发现的Sak的20个NH2末端氨基酸缺乏二级结构，表明Sak的NH2末端区域对于与纤溶酶/纤溶酶原的结合不是必需的，但在这个柔性区域对应于氨基酸11 - 到16的最终或倒数第二个NH2末端位置的带正电荷氨基酸参与纤溶酶分子活性位点的重新配置，使其具有纤溶酶原激活潜力。
