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Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins
Authors:Lowey Susan  Saraswat Lakshmi D  Liu HongJun  Volkmann Niels  Hanein Dorit
Institution:Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA. lowey@physiology.med.uvm.edu
Abstract:The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.
Keywords:cryoEM  electron cryomicroscopy  SH3  src-homology 3 domain  SH1  reactive thiol in myosin heavy chain  ELC  essential light chain  LC1 or A1  the isoform of ELC containing the N-terminal extension of 41 residues  A2  the smaller isoform of ELC that has eight unique residues at the N terminus  but shares the remaining 140 residues with the A1  S1  myosin subfragment-1  1  5-IAEDANS  N-iodoacetyl-N′-(5-sulfo-1-napthyl)ethylenediamine  DDPM  N-(4-dimethylamino-3  5-dinitrophenyl)maleimide  DABM  (4-dimethylaminophenylazophenyl-4′maleimide)  IAF  5-(iodoacetamido) fluorescein  WT  wild-type  FRET  fluorescence resonance energy transfer  IHRSR  iterative helical real space reconstruction
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