Kinetic Analysis Reveals Differences in the Binding Mechanism of Calmodulin and Calmodulin-like Protein to the IQ Motifs of Myosin-10
Abstract
Myo10 is an unconventional myosin with important functions in filopodial motility, cell migration, and cell adhesion. The neck region of Myo10 contains three IQ motifs that bind calmodulin (CaM) or the tissue-restricted calmodulin-like protein (CLP) as light chains. However, little is known about the mechanism of light chain binding to the IQ motifs in Myo10. Binding of CaM and CLP to each IQ motif was assessed by nondenaturing gel electrophoresis and by stopped-flow experiments using fluorescence-labeled CaM and CLP. Although the binding kinetics are different in each case, there are similarities in the mechanism of binding of CaM and CLP to IQ1 and IQ2: for both IQ motifs Ca2+ increased the binding affinity, mainly by increasing the rate of the forward steps. The general kinetic mechanism comprises a two-step process, which in some cases may involve the binding of a second IQ motif with lower affinity. For IQ3, however, the kinetics of CaM binding is very different from that of CLP. In both cases, binding in the absence of Ca2+ is poor, and addition of Ca2+ decreases the Kd to below 10 nM. However, while the CaM binding kinetics are complex and best fitted by a multistep model, binding of CLP is fitted by a relatively simple two-step model. The results show that, in keeping with growing structural evidence, complexes between CaM or CaM-like myosin light chains and IQ motifs are highly diverse and depend on the specific sequence of the particular IQ motif as well as the light chain.
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