title: The unique insert in myosin VI is a structural calcium-calmodulin binding site.
abstract: Myosin VI contains an inserted sequence that is unique among myosin superfamily members and has been suggested to be a determinant of the reverse directionality and unusual motility of the motor. It is thought that each head of a two-headed myosin VI molecule binds one calmodulin (CaM) by means of a single "IQ motif". Using truncations of the myosin VI protein and electrospray ionization(ESI)-MS, we demonstrate that in fact each myosin VI head binds two CaMs. One CaM binds to a conventional IQ motif either with or without calcium and likely plays a regulatory role when calcium binds to its N-terminal lobe. The second CaM binds to a unique insertion between the converter region and IQ motif. This unusual CaM-binding site normally binds CaM with four Ca2+ and can bind only if the C-terminal lobe of CaM is occupied by calcium. Regions of the MD outside of the insert peptide contribute to the Ca(2+)-CaM binding, as truncations that eliminate elements of the MD alter CaM binding and allow calcium dissociation. We suggest that the Ca(2+)-CaM bound to the unique insert represents a structural CaM, and not a calcium sensor or regulatory component of the motor. This structure is likely an integral part of the myosin VI "converter" region and repositions the myosin VI "lever arm" to allow reverse direction (minus-end) motility on actin.
authors.affiliation: Structural Motility, Institut Curie Centre National de la Recherche Scientifique, Unité Mixte de Recherche 144, 26 Rue d'Ulm, 75248 Paris 05, France.
authors.forename: Amel
authors.initials: A
authors.lastname: Bahloul
authors.forename: Guillaume
authors.initials: G
authors.lastname: Chevreux
authors.forename: Amber L
authors.initials: AL
authors.lastname: Wells
authors.forename: Davy
authors.initials: D
authors.lastname: Martin
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Nolt
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Yang
authors.forename: Li-Qiong
authors.initials: LQ
authors.lastname: Chen
authors.forename: Noëlle
authors.initials: N
authors.lastname: Potier
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Steve
authors.initials: S
authors.lastname: Rosenfeld
authors.forename: Anne
authors.initials: A
authors.lastname: Houdusse
authors.forename: H Lee
authors.initials: HL
authors.lastname: Sweeney
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D002147:Calmodulin; C089785:myosin VI; D018995:Myosin Heavy Chains; D002118:Calcium