title: The cargo-binding domain regulates structure and activity of myosin 5.
abstract: Myosin 5 is a two-headed motor protein that moves cargoes along actin filaments. Its tail ends in paired globular tail domains (GTDs) thought to bind cargo. At nanomolar calcium levels, actin-activated ATPase is low and the molecule is folded. Micromolar calcium concentrations activate ATPase and the molecule unfolds. Here we describe the structure of folded myosin and the GTD's role in regulating activity. Electron microscopy shows that the two heads lie either side of the tail, contacting the GTDs at a lobe of the motor domain (approximately Pro 117-Pro 137) that contains conserved acidic side chains, suggesting ionic interactions between motor domain and GTD. Myosin 5 heavy meromyosin, a constitutively active fragment lacking the GTDs, is inhibited and folded by a dimeric GST-GTD fusion protein. Motility assays reveal that at nanomolar calcium levels heavy meromyosin moves robustly on actin filaments whereas few myosins bind or move. These results combine to show that with no cargo, the GTDs bind in an intramolecular manner to the motor domains, producing an inhibited and compact structure that binds weakly to actin and allows the molecule to recycle towards new cargoes.
authors.affiliation: Institute of Molecular and Cellular Biology, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Kavitha
authors.initials: K
authors.lastname: Thirumurugan
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sakamoto
authors.forename: John A
authors.initials: JA
authors.lastname: Hammer
authors.forename: James R
authors.initials: JR
authors.lastname: Sellers
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Knight
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D008854:Microscopy, Electron; D008958:Models, Molecular; D024701:Myosin Type V / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D000199:Actins; D024701:Myosin Type V