Jean-Jacques Schaer

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

Recently, Mills and Johnson [7] and our group [9] provided evidence that calmodulin contains, in addition to the four Ca2+-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 Ca2+ 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 Zn2+ and Mn2+ as selective probes for the auxiliary sites and with Cd2+ as a probe for both types of sites. In the absence of other divalent cations, 6 mol of Zn2+ bind to calmodulin with an identical affinity constant of 2,850 M-1 and a delta H0 of 106 kJ/mol calmodulin. In the presence of millimolar free Ca2+ calmodulin binds, in addition to four Ca2+, six Zn2+ with an affinity constant of 1,200 M-1 and a delta H0 of 47 kJ/mol calmodulin. The Zn2+-Ca2+ antagonism is governed by negative free energy coupling between the capital and auxiliary sites. In contrast, the Zn2+-Mg2+ antagonism follows the rule of straight competition at all six auxiliary sites. Mn2+ also binds exclusively to the auxiliary sites with affinity constants of 800 or 280 M-1 and delta H0 of 45 or 46 kJ/mol calmodulin in the absence and presence of saturating [Ca2+], respectively. Cd2+ binds to the capital sites with an affinity constant of 3.4 10(4) M-1 (delta H = 35 kJ/mol calmodulin) and to the auxiliary sites with ca. 100-fold lower affinity. The Zn2+ much greater than Mn2+ greater than or equal to Cd2+ greater than Mg2+ 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.

Thermodynamics of the binding of calcium and strontium to bovine α-lactalbumin

Microcalorimetry and equilibrium gel filtration were used to determine the thermodynamic functions ΔH°, ΔG° and ΔS° guiding the interaction of Ca2+ and Sr2+ with bovine α‐lactalbumin. Two methods of nearly complete metal removal from the protein gave identical results. The single Ca‐ and Sr‐binding site, which has moderate affinity for these ions (K ca = 2.5 × 106 M−1 and K Sr = 5.1 × 105 M−1), displays unusually large enthalpy changes of −118 kJ · mol−1 for Ca2+ and −75 kJ·mol−1 for Sr2+. The concomitant reaction entropies equal −273 and −142 J·K−1· mol−1, respectively.

Cation Binding to Calmodulin and Relation to Function

Calmodulin (CaM) is intimately involved in the stimulus-response coupling in eukaryotic cells since it is the prime sensor of transient increases of the free intracellular [Ca2+] and conveys the signal to multiple target enzymes, especially protein kinases and phosphatases. For a general survey of the properties of CaM, the reader is referred to Cheung (1980), Klee and Vanaman (1982), Manalan and Klee (1984) and Wang et al. (1985) (see also Cheung this Vol.). This paper deals more specifically with the mode of action of CaM. Although in the last decade our understanding of its action has considerably increased, some enigmas remain and, unfortunately, some basic and long-lasting controversies on its mode of action have not been solved yet. In this paper, two of these are examined: (a) cation binding to CaM, and (b) the thermodynamics of the interaction of CaM with its targets.