title: Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels.
abstract: Actin and myosin are the two main proteins required for cell motility and muscle contraction. The structure of their strongly bound complex-rigor state-is a key for delineating the functional mechanism of actomyosin motor. Current knowledge of that complex is based on models obtained from the docking of known atomic structures of actin and myosin subfragment 1 (S1; the head and neck region of myosin) into low-resolution electron microscopy electron density maps, which precludes atomic- or side-chain-level information. Here, we use radiolytic protein footprinting for global mapping of sites across the actin molecules that are impacted directly or allosterically by myosin binding to actin filaments. Fluorescence and electron paramagnetic resonance spectroscopies and cysteine actin mutants are used for independent, residue-specific probing of S1 effects on two structural elements of actin. We identify actin residue candidates involved in S1 binding and provide experimental evidence to discriminate between the regions of hydrophobic and electrostatic interactions. Focusing on the role of the DNase I binding loop (D-loop) and the W-loop residues of actin in their interactions with S1, we found that the emission properties of acrylodan and the mobility of electron paramagnetic resonance spin labels attached to cysteine mutants of these residues change strongly and in a residue-specific manner upon S1 binding, consistent with the recently proposed direct contacts of these loops with S1. As documented in this study, the direct and indirect changes on actin induced by myosin are more extensive than known until now and attest to the importance of actin dynamics to actomyosin function.
authors: Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90095, USA.
authors: Zeynep A
authors: ZA
authors: Oztug Durer
authors: J K Amisha
authors: JK
authors: Kamal
authors: Sabrina
authors: S
authors: Benchaar
authors: Mark R
authors: MR
authors: Chance
authors: Emil
authors: E
authors: Reisler
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes; D017665:Hydroxyl Radical / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism*
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D017665:Hydroxyl Radical; D009218:Myosins