Regulatory implications of a novel mode of interaction of calmodulin with a double IQ-motif target sequence from murine dilute myosin V.

Apo-Calmodulin acts as the light chain for unconventional myosin V, and treatment with Ca2+ can cause dissociation of calmodulin from the 6IQ region of the myosin heavy chain. The effects of Ca2+ on the stoichiometry and affinity of interactions of calmodulin and its two domains with two myosin-V peptides (IQ3 and IQ4) have therefore been quantified in vitro, using fluorescence and near- and far-UV CID. The results with separate domains show their differential affinity in interactions with the IQ motif, with the apo-N domain interacting surprisingly weakly. Contrary to expectations, the effect of Ca2+ on the interactions of either peptide with either isolated domain is to increase affinity, reducing the K-d at physiological ionic strengths by >200-fold to similar to75 nM for the N domain, and similar to10-fold to similar to15 nM for the C domain. Under suitable conditions, intact (holo- or apo-) calmodulin can bind up to two IQ-target sequences. Interactions of apo- and holo-calmodulin with the double-length, concatenated sequence (IQ34) can result in complex stoichiometries. Strikingly, holo-calmodulin forms a high-affinity 1:1 complex with IQ34 in a novel mode of interaction, as a bridged structure wherein two calmodulin domains interact with adjacent IQ motifs. This apparently imposes a steric requirement for the alpha-helical target sequence to be discontinuous, possibly in the central region, and a model structure is illustrated. Such a mode of interaction could account for the Ca2+-dependent regulation of myosin V in vitro motility, by changing the structure of the regulatory complex, and paradoxically causing calmodulin dissociation through a change in stoichiometry, rather than a Ca2+-dependent reduction in affinity.