Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins.
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.
Lowey
Susan
S
Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA. lowey@physiology.med.uvm.edu
Saraswat
Lakshmi D
LD
Liu
HongJun
H
Volkmann
Niels
N
Hanein
Dorit
D
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D020285:Cryoelectron Microscopy; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009218:Myosins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D018909:src Homology Domains
D020033:Protein Isoforms; D009218:Myosins