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 Ca(2+) can cause dissociation of calmodulin from the 6IQ region of the myosin heavy chain. The effects of Ca(2+) 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 Ca(2+) 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 approximately 75 nM for the N domain, and approximately 10-fold to approximately 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 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 Ca(2+)-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 Ca(2+)-dependent reduction in affinity.
Martin
Stephen R
SR
Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
Bayley
Peter M
PM
D020816:Amino Acid Motifs; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000378:metabolism*; D024701:Myosin Type V / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
D001059:Apoproteins; D002147:Calmodulin; D029721:Drosophila Proteins; C513082:Myo5a protein, mouse; D010446:Peptide Fragments; D024701:Myosin Type V; D018995:Myosin Heavy Chains; D002118:Calcium