Zinc Induces Conformational Changes in Domain 5 [K420‐S513] of High Molecular Mass Kininogen (HK‐D5)

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Domain 5 (D5) of HK contains 23 histidine residues putatively involved in the binding of metals to cells and stabilization of the D5 molecule. Recombinant HK-D5 shows zinc-induced increases in apparent molecular mass using surface plasmon resonance (SPR) with D5 amine coupled to a CM5 chip. Addition of Zn2+ yields an increase in SPR signal (RU) attributed to conformational changes in the D5 molecule (the mass of ionic Zinc is below SPR detection levels). A plot of RU vs. [Zn2+] increases steeply between 1 and 10 uM Zn2+ and reaches a plateau at ~200 uM. Parallel experiments with Ca2+ and Mn2+ exhibit no apparent mass changes whereas Co2+ also shows evidence for conformational changes. Recombinant peptides K420-D474 and H475-K502 showed similar behavior to the parent D5; maximal RU values are proportional to the number of His in each fragment, confirming His involvement in ligand binding. Gel filtration profiles of D5 shows a second peak with reduced mobility upon addition of 100 uM Zn2+. CD and NMR spectra of D5 in the absence and presence of metal ions are consistent with a zinc-induced transition from a largely unstructured conformation to a dynamic structure enriched in beta-sheet. The zinc-complex also shows enhanced tryptophan fluorescence due to burial of W501. Thus, zinc binding to HK-D5 is accompanied by dramatic structural changes and involves many of its His side chains. Since the interactions of HK with platelets, leukocytes and endothelial cells all require zinc, the findings have implications with respect to hemostasis, inflammation and angiogenesis. Supported by CA 23121