The human α2-plasmin inhibitor: functional characterization of the unique plasmin(ogen)-binding region

The human α2-plasmin inhibitor (A2PI) possesses unique N- and C-terminal extensions that significantly influence its biological activities. The C-terminal segment, A2PIC (Asn398-Lys452), contains six lysines thought to be involved in the binding to lysine-binding sites in the kringle domains of human plasminogen, of which four (Lys422, Lys429, Lys436, Lys452) are completely and two (Lys406, Lys415) 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 Lys452 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 Lys452 and internal Lys436 of rA2PIC and isolated K1+2+3, whereas the other internal lysine residues contribute only to a minor extent to the binding process. Sulfated Tyr445 in the unique C-terminal segment revealed no influence on the binding affinity to kringle domain