Zinc-dependent conformational changes in domain D5 of high molecular mass kininogen modulate contact activation.
Human high molecular mass kininogen (HK) participates as nonenzymatic cofactor in the contact system. Here, we show that recombinant domain D5 of HK (rD5) prolongs the clotting time of the intrinsic pathway of coagulation and attenuates the generation of bradykinin. Further studies indicate that a correct fold of domain D5 within HK is required for the activation of the contact system. The folding of rD5 seems to be modulated by the metal ions Zn2+, Ni2+, and Cu2+ as a specific antibody directed against the zinc-binding site in HK binds to HK and rD5 in a metal ion concentration dependent manner. The finding that these three metal ions specifically affect contact activation suggests that they regulate the accessibility of rD5 for negatively charged surfaces. Support for the assumption that the observed phenomena are due to conformational changes was obtained by fluorescence spectroscopy of rD5, demonstrating that its fluorescence spectrum was changed in the presence of ZnCl2. Moreover, negative staining electron microscopy experiments suggest that the zinc-induced changes in D5 also affect the conformation of the entire HK protein. The present data emphasize the role of zinc and other metal ions in the regulation of contact activation.
Herwald
H
H
Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, Tornavägen 10, S-221 Lund, Sweden. Heiko.Herwald@medkem.lu.se
Mörgelin
M
M
Svensson
H G
HG
Sjöbring
U
U
D000595:Amino Acid Sequence; D001920:Bradykinin / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D019679:Kininogen, High-Molecular-Weight / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D014914:Whole Blood Coagulation Time; D015032:Zinc / Q000494:pharmacology*
D019679:Kininogen, High-Molecular-Weight; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015032:Zinc; D001920:Bradykinin