Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

BACKGROUND: Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. RESULTS: In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with K(d )values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca(2+ )substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail) each bound 2–3 calmodulin molecules. At a physiologically relevant temperature (25°C) and under low-Ca(2+ )conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 μM free calmodulin. Ca(2+ )dissociated nearly all calmodulins from T701-Myo1c at 25°C; one calmodulin was retained if 5 μM free calmodulin was present. CONCLUSIONS: We inferred from these data that at 25°C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca(2+ )is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca(2+ )elevation, all calmodulin except that bound to IQ3 should dissociate.