METAL-ION BINDING TO DOG OSTEOCALCIN STUDIED BY H-1-NMR SPECTROSCOPY

One-dimensional H-1 NMR was employed to study the effects of Ca2+ and Lu3+ binding on the apo and calcium-saturated forms of dog bone Gla pr otein (BGP, osteocalcin). Titration of apo dog BGP with Ca2+ in 20 mM NaCl showed spectral perturbations consistent with the binding of 5 mo l equiv of calcium in the NMR slow-exchange limit. The first 2 Ca2+ eq uiv induced significant conformational changes in the apoprotein, bind ing cooperatively with a K(d1) approximately 5.0 x 10(-4) M and a Hill coefficient H = 2.3 in 20 mM NaCl. The last 3 equiv bound with a slig htly weaker affinity and did not induce significant structural changes . Neither the affinity nor the stoichiometry of calcium binding was si gnificantly altered at 150 mM NaCl. The addition of only 1 Lu3+ equiv to apo dog osteocalcin was sufficient to induce the same spectral pert urbations as 2 Ca2+ ions. The addition of 2 Lu3+ equiv to calcium-satu rated osteocalcin had little effect on its H-1 NMR spectrum, and BGP a ggregated at [Lu3+]o/[BGP]o ratios greater than 2 in either the presen ce or absence of calcium. The spectrum of calcium-saturated osteocalci n was invariant at less-than-or-equal-to 55-degrees-C (less-than-or-eq ual-to 50-degrees-C in 150 mM NaCl), after which the proton resonances shifted to frequencies more characteristic of apo BGP. Saturation wit h calcium somewhat stabilized the apo dog osteocalcin protein against conformational changes induced at pH extremes; apo BGP was stable at 6 .0 less-than-or-equal-to pH less-than-or-equal-to 10, and calcium-satu rated BGP was stable at 5.8 less-than-or-equal-to pH less-than-or-equa l-to 10. Both our NMR and gel filtration data indicate that calcium-sa turated osteocalcin exists as a dimer at both high and low protein con centrations. A conformational change in dog osteocalcin was thus induc ed by the cooperative association of Ca2+ to two high-affinity sites o n the protein and stabilized by the association of 3 additional Ca2+ e quiv. The results of our temperature and calcium binding studies were consistent with an estimated K(d1) approximately 5.0 x 10(-4) M for th e two high-affinity sites. Lutetium induced the same structural change s in osteocalcin as calcium, but the two high-affinity Ca2+ binding si tes did not have equal affinities for Lu3+. The BGP:Ca2+ complex was u nstable at the low pH conditions induced by osteoclasts during bone re sorption, yet the osteocalcin protein retained a BGP: Ca2+-like confor mation at low pH. However, unlike the calcium-saturated form of the pr otein, osteocalcin was monomeric at low pH.