Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.
Cell Mol Life Sci. 2010 May;67(9):1505-18. doi: 10.1007/s00018-010-0264-3. Epub 2010 Jan 29.
The human alpha(2)-plasmin inhibitor (A2PI) possesses unique N- and C-terminal extensions that significantly influence its biological activities. The C-terminal segment, A2PIC (Asn(398)-Lys(452)), contains six lysines thought to be involved in the binding to lysine-binding sites in the kringle domains of human plasminogen, of which four (Lys(422), Lys(429), Lys(436), Lys(452)) are completely and two (Lys(406), Lys(415)) are partially conserved. Multiple Lys to Ala mutants of A2PIC were expressed in Escherichia coli and used in intrinsic fluorescence titrations with kringle domains K1, K4, K4 + 5, and K1 + 2 + 3 of human plasminogen. We were able to identify the C-terminal Lys(452) as the main binding partner in recombinant A2PIC (rA2PIC) constructs with isolated kringles. We could show a cooperative, zipper-like enhancement of the interaction between C-terminal Lys(452) and internal Lys(436) of rA2PIC and isolated K1 + 2 + 3, whereas the other internal lysine residues contribute only to a minor extent to the binding process. Sulfated Tyr(445) in the unique C-terminal segment revealed no influence on the binding affinity to kringle domains.
人α(2)-纤溶酶抑制剂 (A2PI) 具有独特的 N-末端和 C-末端延伸，这显著影响其生物学活性。C-末端片段 A2PIC（Asn(398)-Lys(452)）含有六个赖氨酸，据认为这些赖氨酸参与与人类纤溶酶原kringle 结构域中的赖氨酸结合位点结合，其中四个（Lys(422)、Lys(429)、Lys(436)、Lys(452)）完全保守，两个（Lys(406)、Lys(415)）部分保守。在大肠杆菌中表达了 A2PIC 的多个 Lys 到 Ala 突变体，并用于与人类纤溶酶原kringle 结构域 K1、K4、K4 + 5 和 K1 + 2 + 3 的固有荧光滴定。我们能够鉴定出 C-末端 Lys(452)是与分离的 kringles 结合的重组 A2PIC(rA2PIC)构建体中的主要结合伴侣。我们可以显示出 C-末端 Lys(452)与 rA2PIC 和分离的 K1 + 2 + 3 之间的拉链式协同增强相互作用，而其他内部赖氨酸残基仅对结合过程有较小的贡献。独特的 C-末端片段中的硫酸化 Tyr(445)对与 kringle 结构域的结合亲和力没有影响。
