High affinity Ca2+ binding sites of calmodulin are critical for the regulation of myosin Ibeta motor function
Department of Physiology
Actins; Adenosine Triphosphatases; Animals; Binding Sites; Calcium; Calcium-Binding Proteins; Calmodulin; Calmodulin-Binding Proteins; Cattle; Models, Molecular; Mutation; Myosin Heavy Chains; Protein Binding; Protein Conformation; Recombinant Proteins
Life Sciences | Medicine and Health Sciences
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.
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Citation: J Biol Chem. 1998 Aug 7;273(32):20481-6.
The Journal of biological chemistry
Zhu, Tong; Beckingham, Kathleen M.; and Ikebe, Mitsuo, "High affinity Ca2+ binding sites of calmodulin are critical for the regulation of myosin Ibeta motor function" (1998). Open Access Articles. 778.