Evidence for four capital and six auxiliary cation-binding sites on calmodulin: Divalent cation interactions monitored by direct binding and microcalorimetry

Abstract

Recently, Mills and Johnson [7] and our group [9] provided evidence that calmodulin contains, in addition to the four Ca 2+-binding sites (capital sites), which are essential for drug- and enzyme-binding, a number of divalent cation-binding sites of different ion selectivity (auxiliary sites), which modulate drug-binding as well as the affinity of Ca 2+ for the capital sites. In the present study, the number of auxiliary sites and their relationship to the capital sites were determined by equilibrium gel filtration and by flow microcalorimetry with Zn 2+ and Mn 2+ as selective probes for the auxiliary sites and with Cd 2+ as a probe for both types of sites. In the absence of other divalent cations, 6 mol of Zn 2+ bind to calmodulin with an identical affinity constant of 2,850 M −1 and a ΔH 0 of 106 kJ/mol calmodulin. In the presence of millimolar free Ca 2+ calmodulin binds, in addition to four Ca 2+, six Zn 2+ with an affinity constant of 1,200 M −1 and a ΔH 0 of 47 kJ/mol calmodulin. The Zn 2+-Ca 2+ antagonism is governed by negative free energy coupling between the capital and auxiliary sites. In contrast, the Zn 2+-Mg 2+ antagonism follows the rule of straight competition at all six auxiliary sites. Mn 2+ also binds exclusively to the auxiliary sites with affinity constants of 800 or 280 M −1 and ΔH 0 of 45 or 46 kJ/mol calmodulin in the absence and presence of saturating [Ca 2+], respectively. Cd 2+ binds to the capital sites with an affinity constant of 3.4 10 4 M −1 (ΔH = 35 kJ/mol calmodulin) and to the auxiliary sites with ca. 100-fold lower affinity. The Zn 2+ > Mn 2+ ≥ Cd 2+ > Mg 2+ selectivity of the auxiliary sites corroborates the potencies of these cations in modulating drug binding. The auxiliary site-specific cations are unable to promote high-affinity complex formation between calmodulin and melittin.