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J Biol Chem, Vol. 274, Issue 49, 35159-35171, December 3, 1999

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

Wei-Lih Lee^§, E. Michael Ostap^¶, Henry G. Zot, and Thomas D. Pollard^§

From the ^§ Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, the  Department of Biology, Eastern Michigan University, Ypsilanti, Michigan 48197, the  BCMB Graduate Program, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the ^¶ Pennsylvania Muscle Institute and the Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

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 -chymotrypsin digestion. The peptide link between TH-1 and TH-2/3 is cleaved by trypsin, -chymotrypsin, and endo-AspN but not V8 protease. The CD spectra of TH-2/3 indicate predominantly random structure, turns, and -strands but no -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.

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