Positive cooperative binding of calcium to bovine brain calmodulin

Abstract

Equilibrium dialysis measurements of the binding of Ca2+ to calmodulin have confirmed the existence of four high affinity Ca2+-binding sites (Kd between 3 X 10(-6) and 2 X 10(-5) M). In the presence of 3 mM Mg2+, the dissociation constants for Ca2+ are increased two- to fourfold (Kd between 5 X 10(-6) and 4 X 10(-5) M). Positive cooperativity of Ca2+ binding was observed at low Ca2+ concentrations with Hill coefficients of 1.33 and 1.22 in the absence and presence of 3 mM Mg2+, respectively. The positive cooperativity is compatible with the steepness of the Ca2+ dependence of the conformational transition associated with the binding of 2 mol of Ca2+/mol of calmodulin. This conformational change, which affects the environment of the aromatic residues of calmodulin as measured by UV absorption and near-UV circular dichroism spectroscopy, is not the result of a monomer-dimer equilibrium mediated by Ca2+. Binding of Ca2+ to calmodulin is believed to occur by a sequential mechanism generating at least four different conformers of the protein and its free and liganded states. Even though the major conformational change is almost complete upon binding of 2 mol of Ca2+/mol of calmodulin, the activation of cyclic nucleotide phosphodiesterase measured in the presence of limiting concentrations of calmodulin suggests that a calmodulin Ca3-42+ complex is required for interaction of calmodulin with the enzyme. As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex (Kd = 1-3 X 10(-9) M), the Ca2+ dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold of Ca2+ concentration for control of enzyme activity.

A 113Cd NMR study of calmodulin and its interaction with calcium, magnesium and trifluoperazine

“…Since the ~t~sities of the "3Cd NMR signal A and B are observed to increase in parallel as '13CdZ+ is successively added to calcium free calmodulin, this indicates that the individual high afftity binding constants, K& and K& are almost identical or that the 2 high affinity sites display positive cooperativity [26]. The same type of behaviour is observed for the 2 high affinity sites of rabbit skeletal muscle TnC P51.…”

An allosteric model of calmodulin explains differential activation of PP2B and CaMKII

“…Many investigators measuring calcium binding to calmodulin (16,18,28,42) have used an Adair function (43), or a modified version thereof, to describe calmodulin binding to calcium. This approach allows for different microscopic affinities for the four binding events and thus for detailed models of calcium binding to calmodulin.…”

“…Each of these models reflects some properties of calmodulin and is reasonably applicable in contexts in which only these properties are relevant. However, none of these models can satisfactorily account for all of the observed properties of calmodulin such as cooperativity of calcium binding and different affinities for different calcium-binding sites (16), activation of targets by unsaturated calmodulin (17,18), and increased affinity for calcium upon binding to targets (18)(19)(20). We propose an alternative model, based on a biophysical description of the conformational transitions.…”

Quantitative measurements of the cooperativity in an EF‐hand protein with sequential calcium binding

“…Structural studies have shown that EF-hands are most often arranged in pairs, or higher order assemblies, with a short b-type interaction between the two 12-residue Caz+-binding loops. Pairs of EF-hands, either isolated or in the context of a larger globular domain, have repeatedly been found to bind Ca2+ ions with positive cooperativity, as for example in parvalbumin (Cave et al, 1979;Moeschler et al, 1980), troponin C (Grabarek et al, 1983;Teleman et al, 1983;Pearlstone et al, 1992), calmodulin (Crouch & Klee, 1980;Linse et al, 1990), calbindin Dgk , recoverin (Zozulya & Stryer, 1992), calerythrin (Bylsma et al, 1992), and sarcoplasmic calcium binding protein (Cox & Stein, 1981). However, gaining an understanding of the molecular mechanism underlying the observed cooperativity is an elaborate task involving detailed characterization of all ion ligation states.…”