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

Abstract

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 CD. 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 ∼75 nM for the N domain, and ∼10‐fold to ∼15 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 α‐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.