Identification and chemical synthesis of a substrate-binding site for factor IX on coagulation factor XIa.
We have previously used monoclonal antibodies to identify an epitope on the heavy chain of factor XIa that is a substrate-binding site for factor IX (Sinha, D., Seaman, F.S., and Walsh, P.N. (1987) Biochemistry 26, 3768-3775; Baglia, F.A., Sinha, D., and Walsh, P.N. (1989) Blood 74, 244-251). To define the factor XIa domain that binds factor IX, we have now screened a panel of factor XI heavy chain-derived synthetic peptides for their capacity to inhibit the formation of an activation peptide reflecting factor IX activation by factor XIa. Peptide Asn145-Ala176 (which is located in the second tandem repeat or A2 domain of the factor XI heavy chain) is a competitive inhibitor of factor IX activation by factor XIa with a Ki of 30 nM, whereas structurally similar peptides in the A1, A3, and A4 domains were required at 10-1000-fold higher concentrations for similar effects, and a synthetic peptide identical with a highly homologous region of the heavy chain A2 domain of prekallikrein (Tyr143-Ala176) had no effect on factor IX activation by factor XIa. Because detailed structural information is lacking, a potential three-dimensional structure for the factor XI A2 domain was calculated based on its sequence information in conjunction with previously determined structural constraints. The resulting structure depicted three juxtaposed beta-stranded stem-loops that, based on biological information, constitute a candidate surface for contact with factor IX. The A2 model was therefore used as a template in the rational design of three synthetic peptides (Ala134-Ile146 (peptide a), Leu148-Arg159 (peptide b), and Ile160-Leu172 (peptide c]. When peptides a and b or a and c were added together and the activation of factor IX by factor XIa was examined, a synergistic inhibitory effect was observed, compared with each peptide added individually, whereas peptides b and c showed additive effects. Our data suggest that the sequence of amino acids from Ala134 through Leu172 of the heavy chain of factor XI contains three antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of factor IX.
Baglia
F A
FA
Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Jameson
B A
BA
Walsh
P N
PN
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D005164:Factor IX / Q000378:metabolism*; D015945:Factor XIa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D016339:Spectrometry, Mass, Fast Atom Bombardment
D010455:Peptides; D005164:Factor IX; D015945:Factor XIa