The human alpha(2)-plasmin inhibitor: functional characterization of the unique plasmin(ogen)-binding region.
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.
Gerber
Simon S
SS
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.
Lejon
Sofia
S
Locher
Michael
M
Schaller
Johann
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005341:Fibrinolysin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018082:Kringles; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000979:alpha-2-Antiplasmin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D010455:Peptides; D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin; D010958:Plasminogen; D005341:Fibrinolysin; D008239:Lysine