Mapping of the discontinuous H-kininogen binding site of plasma prekallikrein. Evidence for a critical role of apple domain-2.
Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K(D) = 1.2 x 10(-8) M). Previous work had demonstrated that a discontinuous kininogen-binding site is formed by a proximal part located in A1, a distal part exposed by A4, and other yet unidentified portion(s) of the kallikrein heavy chain. To detect relevant binding segment(s) we recombinantly expressed single apple domains and found a rank order of binding affinity for kininogen of A2 > A4 approximately A1 > A3. Removal of single apple domains in prekallikrein deletion mutants reduced kininogen binding by 21 (A1), 64 (A2), and 24% (A4), respectively, whereas deletion of A3 was without effect. Transposition of homologous A2 domain from prekallikrein to factor XI conferred high-affinity kininogen binding from the former to the latter. The principal role of A2 for H-kininogen docking to the prekallikrein heavy chain was further substantiated by the finding that cleavage of a single peptide bond in A2 drastically diminished the H-kininogen binding affinity. Furthermore, the epitope of monoclonal antibody PKH6 which blocks kallikrein-kininogen complex formation with an IC(50) of 8 nM mapped to the center portion of domain A2. Our data indicate that domain A2 and two flanking sequence segments of A1 and A4 form a discontinuous binding platform for H-kininogen on the prekallikrein heavy chain. Domain-specific antibodies directed to these critical sites efficiently interfered with contact phase-induced bradykinin release from H-kininogen.
Renné
T
T
Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University at Mainz, Duesbergweg 6, D-55099 Mainz, Germany.
Dedio
J
J
Meijers
J C
JC
Chung
D
D
Müller-Esterl
W
W
D001665:Binding Sites; D006801:Humans; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism*; D011288:Prekallikrein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017384:Sequence Deletion
D019679:Kininogen, High-Molecular-Weight; D011288:Prekallikrein