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

J Biol Chem. 1999 Dec 3;274(49):35159-71. doi: 10.1074/jbc.274.49.35159.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acanthamoeba / chemistry*
  • Acanthamoeba / genetics
  • Actins / metabolism*
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Carrier Proteins / metabolism
  • Chromatography, Gel
  • Circular Dichroism
  • Cloning, Molecular
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Myosin Heavy Chains / chemistry*
  • Myosin Heavy Chains / genetics
  • Myosin Heavy Chains / isolation & purification
  • Myosin Heavy Chains / metabolism*
  • Myosins / chemistry*
  • Myosins / genetics
  • Myosins / isolation & purification
  • Myosins / metabolism*
  • Phylogeny
  • Protozoan Proteins / metabolism
  • Recombinant Proteins / metabolism
  • Ultracentrifugation
  • src Homology Domains

Substances

  • Acan125 protein, Acanthamoeba
  • Actins
  • Carrier Proteins
  • DNA, Complementary
  • Protozoan Proteins
  • Recombinant Proteins
  • Myosin Heavy Chains
  • Myosins