title: Organization and ligand binding properties of the tail of Acanthamoeba myosin-IA. Identification of an actin-binding site in the basic (tail homology-1) domain.
abstract: The Acanthamoeba myosin-IA heavy chain gene encodes a 134-kDa protein with a catalytic domain, three potential light chain binding sites, and a tail with separately folded tail homology (TH) -1, -2, and -3 domains. TH-1 is highly resistant to trypsin digestion despite consisting of 15% lysine and arginine. TH-2/3 is resistant to alpha-chymotrypsin digestion. The peptide link between TH-1 and TH-2/3 is cleaved by trypsin, alpha-chymotrypsin, and endo-AspN but not V8 protease. The CD spectra of TH-2/3 indicate predominantly random structure, turns, and beta-strands but no alpha-helix. The hydrodynamic properties of TH-2/3 (Stokes' radius of 3.0 nm, sedimentation coefficient of 1.8 S, and molecular mass of 21.6 kDa) indicate that these domains are as long as the whole myosin-I tail in reconstructions of electron micrographs. Furthermore, separately expressed and purified TH-1 binds with high affinity to TH-2/3. Thus we propose that TH-1 and TH-2/3 are arranged side by side in the myosin-IA tail. Separate TH-1, TH-2, and TH-2/3 each binds muscle actin filaments with high affinity. Salt inhibits TH-2/3 binding to muscle actin but not amoeba actin filaments. TH-1 enhances binding of TH-2/3 to muscle actin filaments at physiological salt concentration, indicating that TH-1 and TH-2/3 cooperate in actin binding. An intrinsic fluorescence assay shows that TH-2/3 also binds with high affinity to the protein Acan125 similar to the SH3 domain of myosin-IC. Phylogenetic analysis of SH3 sequences suggests that myosin-I acquired SH3 domain after the divergence of the genes for myosin-I isoforms.
authors.affiliation: BCMB Graduate Program, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: W L
authors.initials: WL
authors.lastname: Lee
authors.forename: E M
authors.initials: EM
authors.lastname: Ostap
authors.forename: H G
authors.initials: HG
authors.lastname: Zot
authors.forename: T D
authors.initials: TD
authors.lastname: Pollard
mesh_terms: D000048:Acanthamoeba / Q000737:chemistry* / Q000235:genetics; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D015800:Protozoan Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014461:Ultracentrifugation; D018909:src Homology Domains
chemical_list: C096372:Acan125 protein, Acanthamoeba; D000199:Actins; D002352:Carrier Proteins; D018076:DNA, Complementary; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D018995:Myosin Heavy Chains; D009218:Myosins