title: The myosin interacting-heads motif is present in the relaxed thick filament of the striated muscle of scorpion.
abstract: Electron microscopy (EM) studies of 2D crystals of smooth muscle myosin molecules have shown that in the inactive state the two heads of a myosin molecule interact asymmetrically forming a myosin interacting-heads motif. This suggested that inactivation of the two heads occurs by blocking of the actin-binding site of one (free head) and the ATP hydrolysis site of the other (blocked head). This motif has been found by EM of isolated negatively stained myosin molecules of unregulated (vertebrate skeletal and cardiac muscle) and regulated (invertebrate striated and vertebrate smooth muscle) myosins, and nonmuscle myosin. The same motif has also been found in 3D-reconstructions of frozen-hydrated (tarantula, Limulus, scallop) and negatively stained (scallop, vertebrate cardiac) isolated thick filaments. We are carrying out studies of isolated thick filaments from other species to assess how general this myosin interacting-heads motif is. Here, using EM, we have visualized isolated, negatively stained thick filaments from scorpion striated muscle. We modified the iterative helical real space reconstruction (IHRSR) method to include filament tilt, and band-pass filtered the aligned segments before averaging, achieving a 3.3 nm resolution 3D-reconstruction. This reconstruction revealed the presence of the myosin interacting-heads motif (adding to evidence that is widely spread), together with 12 subfilaments in the filament backbone. This demonstrates that conventional negative staining and imaging can be used to detect the presence of the myosin interacting-heads motif in helically ordered thick filaments from different species and muscle types, thus avoiding the use of less accessible cryo-EM and low electron-dose procedures.
authors.affiliation: Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas-IVIC, Apdo. 20632, Caracas 1020A, Venezuela. pinto.misle@gmail.com
authors.forename: Antonio
authors.initials: A
authors.lastname: Pinto
authors.forename: Fredi
authors.initials: F
authors.lastname: Sánchez
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Alamo
authors.forename: Raúl
authors.initials: R
authors.lastname: Padrón
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D062105:Molecular Docking Simulation; D057054:Molecular Imaging; D009126:Muscle Relaxation; D054792:Muscle, Striated / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry*; D054730:Protein Interaction Domains and Motifs; D012605:Scorpions / Q000737:chemistry*
chemical_list: D000255:Adenosine Triphosphate; D009218:Myosins