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J Biol Chem, Vol. 273, Issue 32, 20481-20486, August 7, 1998

High Affinity Ca²⁺ Binding Sites of Calmodulin Are Critical for the Regulation of Myosin I Motor Function

Tong Zhu, Kathy Beckingham, and Mitsuo Ikebe

From the Department of Physiology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655 and  Department of Biochemistry and Cell Biology, Rice Univesity, Houston, Texas 77005

We coexpressed myosin I 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 I. 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 I with E12Q calmodulin has the regulatory characteristics similar to myosin I containing wild-type calmodulin, while myosin I with E34Q or E1234Q calmodulin lose all Ca²⁺ regulation. While the increase in myosin I ATPase activity paralleled the dissociation of 1 mol of calmodulin from myosin I 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 I 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.

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