Metal Binding and Metal-Induced Conformational Change of Frog Bone γ-Carboxyglutamic Acid-Containig Protein

Abstract

Ca²⁺ or Cd²⁺ binding and the conformational change induced by the metal binding in two frog bone Gla-proteins (BGP, termed BGP-1 and BGP-2) were studied by equilibrium dialysis and CD measurement. By CD measurement in the far-ultraviolet region, the a-helix content of both apoBGPs was found to be 8%. Binding of both Ca²⁺ and Cd²⁺ was accompanied with a change in the CD spectrum, and the a-helix content increased to 15 and 25% for BGP-1 and BGP-2, respectively. CD measurement in the near-ultraviolet region indicated that the environment of aromatic amino acid residues in the protein molecule was changed by metal binding. Equilibrium dialysis experiments indicated that each of these two protein binds specifically 2mol of Ca²⁺, and nonspecifically an additional 3-4mol of Ca²⁺ in 0.02M Tris-HCl/0.15M NaCl (pH 7.4), at 4°C. According to the two separate binding sites model, BGP-1 has 1 high-affinity Ca²⁺ binding site (K_d1=0.17mM) and 1 low-affinity site (K_d2=0.29mM), and BGP-2 contains 1 high-affinity site (K_d1=0.14mM) and 1 low-affinity site (K_d2=0.67mM). In addition, 2 Cd²⁺ bound to a high-affinity binding site on BGP-1 with K_d1, of 10.4μM, and 1 Cd²⁺ bound to a low-affinity binding site with K_d2, of 41.5μM. On the other hand, BGP-2 had three classes of binding sites and 1 CD²⁺ bound to each binding site with K_d1=3.6μM, K_d2=16.3μM, K_d3=51.7μM, respectively. Parallel analysis of Ca²⁺ binding and CD titration at 222nm with Ca²⁺ revealed that conformational change was maximum at 4.9mM Ca²⁺, that is, sufficient to bind more than 5mol of Ca²⁺ to BGPs. In contrast, cooperativity of Cd²⁺-induced conformational change was observed when the first Cd²⁺ bound to BGP, and maximum CD change occurred at 100μM Cd²⁺, that is, sufficient to bind 2mol of Cd²⁺ to independent binding sites.