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

The Acanthamoeba myosin-LA heavy chain gene encodes a 134-kDa protein with a catalytic domain, three potential light chain binding sites, and a tail w ith separately folded tail homology (TH) -1, -2, and -3 domains. TH-1 is hi ghly resistant to trypsin digestion despite consisting of 15% lysine and ar ginine, TH-2/3 is resistant to alpha-chymotrypsin digestion. The peptide li nk between TH-1 and TH-2/3 is cleaved by trypsin, alpha-chymotrypsin, and e ndo-AspN but not V8 protease, The CD spectra of TH-2/3 indicate predominant ly random structure, turns, and beta-strands but no alpha-helix, The hydrod ynamic properties of TH-2/3 (Stokes' radius of 3.0 nn, sedimentation coeffi cient 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 micr ographs. Furthermore, separately expressed and purified TH-1 binds with hig h affinity to TH-2/3, Thus we propose that TH-1 and TH-2/3 are arranged sid e 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, indicatin g that TH-1 and TH-2/3 cooperate in actin binding. An intrinsic fluorescenc e assay shows that TH-2/3 also binds with high affinity to the protein Acan 125 similar to the SH3 domain of myosin-IC, Phylogenetic analysis of SH3 se quences suggests that myosin-I acquired SH3 domain after the divergence of the genes for myosin-I isoforms.