Research article

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

Peter G Gillespie¹ and Janet L Cyr^1,2

¹Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, Portland OR 97239, USA

²Present address: Department of Otolaryngology & Sensory Neuroscience Research Center, West Virginia University School of Medicine, Morgantown WV 26506, USA

author email corresponding author email

BMC Biochemistry 2002, 3:31doi:10.1186/1471-2091-3-31

Published:	26 November 2002

Abstract

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. Ca²⁺ 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-Ca²⁺ conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 μM free calmodulin. Ca²⁺ 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 Ca²⁺ is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca²⁺ elevation, all calmodulin except that bound to IQ3 should dissociate.