title: The two IQ-motifs and Ca2+/calmodulin regulate the rat myosin 1d ATPase activity.
abstract: The light chain binding domain of rat myosin 1d consists of two IQ-motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca2+/CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d-head, Myo1d-IQ1, Myo1d-IQ1.2, Myo1d-IQ2 and Myo1dDeltaLV-IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca2+. IQ2 had a lower affinity for CaM in the absence of Ca2+ than in the presence of Ca2+. The actin-activated ATPase activity of Myo1d was approximately 75% inhibited by Ca2+-binding to CaM. This inhibition was observed irrespective of whether IQ1, IQ2 or both IQ1 and IQ2 were fused to the head. Based on the measured Ca2+-dependence, we propose that Ca2+-binding to the C-terminal pair of high affinity sites in CaM inhibits the Myo1d actin-activated ATPase activity. This inhibition was due to a conformational change of the C-terminal lobe of CaM remaining bound to the IQ-motif(s). Interestingly, a similar but Ca2+-independent inhibition of Myo1d actin-activated ATPase activity was observed when IQ2, fused directly to the Myo1d-head, was rotated through 200 degrees by the deletion of two amino acids in the lever arm alpha-helix N-terminal to the IQ-motif.
authors.affiliation: Institute for General Zoology and Genetics, Westfälische Wilhelms University, Münster, Germany.
authors.forename: Danny
authors.initials: D
authors.lastname: Köhler
authors.forename: Sandra
authors.initials: S
authors.lastname: Struchholz
authors.forename: Martin
authors.initials: M
authors.lastname: Bähler
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009218:Myosins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D016415:Sequence Alignment
chemical_list: D000199:Actins; D002147:Calmodulin; D009218:Myosins; D002118:Calcium