Mechanism of the Ca(2)+-dependent interaction between S100A4 and tail fragments of nonmuscle myosin heavy chain IIA
Department of Cell Biology
Amino Acid Sequence; Base Sequence; Calcium; DNA Primers; Humans; Kinetics; Microscopy, Electron; Molecular Motor Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutant Proteins; Myosin Heavy Chains; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Protein Interaction Domains and Motifs; Recombinant Proteins; S100 Proteins
The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca(2+) binds to the EF2 domain of S100A4 with micromolar affinity and that the K(d) value for Ca(2+) is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in K(d) results from a reduced dissociation rate constant (from 16 s(-1) to 0.3 s(-1) in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca(2+)]. However, for Ca(2+) transients to be effective in further promoting dissociation, the elevated Ca(2+) signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein-myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data.
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Citation: J Mol Biol. 2011 Jan 28;405(4):1004-26. Epub 2010 Nov 24. Link to article on publisher's site
Journal of molecular biology