Abstract
We coexpressed myosin Ibeta heavy chain with three different calmodulin mutants in which the two Ca2+-binding sites of the two N-terminal domain (E12Q), C-terminal domain (E34Q), or all four sites (E1234Q) are mutated in order to define the importance of these Ca2+ binding sites to the regulation of myosin Ibeta. The calmodulin mutated at the two Ca2+ binding sites in N-terminal domain and C-terminal domain lost its lower affinity Ca2+ binding site and higher affinity Ca2+ binding site, respectively. We found that, based upon the change in the actin-activated ATPase activities and actin translocating activities, myosin Ibeta with E12Q calmodulin has the regulatory characteristics similar to myosin Ibeta containing wild-type calmodulin, while myosin Ibeta with E34Q or E1234Q calmodulin lose all Ca2+ regulation. While the increase in myosin Ibeta ATPase activity paralleled the dissociation of 1 mol of calmodulin from myosin Ibeta heavy chain for both wild type (above pCa 5) and E12Q calmodulin (above pCa 6), the Ca2+ level required for the inhibition of actin-translocating activity of myosin Ibeta was lower than that required for dissociation of calmodulin, suggesting that the conformational change induced by the binding of Ca2+ at the high affinity site but not the dissociation of calmodulin is critical for the inhibition of the motor activity. Our results suggest that the regulation of unconventional myosins by Ca2+ is directly mediated by the Ca2+ binding to calmodulin, and that the C-terminal pair of Ca2+-binding sites are critical for this regulation.
Highlights
Myosins are a large family of molecular motors responsible for diverse forms of cell contractility and motility, such as muscle contraction, chemotaxis, and phagocytosis [1]
All of the calmodulin mutants copurified with myosin I heavy chain, suggesting that the mutations do not affect the binding of calmodulin to myosin I heavy chain
We have coexpressed myosin I heavy chain with three different calmodulin mutants, in which the conserved critical glutamic acid residue at the 12th position of the two N-terminal, two C-terminal, or all four of the Ca2ϩ binding loops were substituted by glutamine
Summary
Myosins are a large family of molecular motors responsible for diverse forms of cell contractility and motility, such as muscle contraction, chemotaxis, and phagocytosis [1]. Myosin I is found in hair bundles purified from the bullfrog sacculus, suggesting that myosin I may function as an adaptation motor which regulates the tip link-associated cation selective channels [11, 12] Studies from both naturally isolated and recombinant myosins I have shown that calmodulin is associated with myosin I heavy chain [5, 13]. Myosin I, like all the other vertebrate unconventional myosins, has several repeats of a 24 –30-amino acid sequence called the IQ motif at the neck region between the myosin head motor domain and the tail domain This motif has been suggested to provide the binding site for EF-hand family proteins such as calmodulin [1, 14]. Mutagenesis studies have shown that a conserved glutamic acid residue at the 12th position of each Ca2ϩ-binding loop is critical for Ca2ϩ binding, and substitution of this conserved glutamic acid with glutamine in each Ca2ϩ-binding site abolishes its Ca2ϩ binding ability [17, 20]
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