Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

BMC Biochem. 2002 Nov 26;3:31. doi: 10.1186/1471-2091-3-31. Epub 2002 Nov 26.

Abstract

Background: Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report.

Results: In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2-4 microM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca2+ substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail) each bound 2-3 calmodulin molecules. At a physiologically relevant temperature (25 degrees C) and under low-Ca2+ conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 microM free calmodulin. Ca2+ dissociated nearly all calmodulins from T701-Myo1c at 25 degrees C; one calmodulin was retained if 5 microM free calmodulin was present.

Conclusions: We inferred from these data that at 25 degrees C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca2+ is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca2+ elevation, all calmodulin except that bound to IQ3 should dissociate.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Calmodulin / chemistry
  • Calmodulin / metabolism*
  • Cattle
  • Hydrazines / metabolism
  • Hydrophobic and Hydrophilic Interactions
  • Molecular Sequence Data
  • Molecular Weight
  • Myosin Type I / chemistry
  • Myosin Type I / metabolism*
  • Peptide Mapping / methods
  • Peptides / chemical synthesis
  • Peptides / chemistry
  • Peptides / metabolism*
  • Protein Binding
  • Rana catesbeiana
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism*
  • Sequence Analysis, Protein / methods

Substances

  • Alexa 488 hydrazide
  • Calmodulin
  • Hydrazines
  • Peptides
  • Recombinant Proteins
  • Myosin Type I