Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides.
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.
Gillespie
Peter G
PG
Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, Portland OR 97239, USA. gillespp@ohsu.edu
Cyr
Janet L
JL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D006834:Hydrazines / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D008970:Molecular Weight; D024461:Myosin Type I / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D020539:Sequence Analysis, Protein / Q000379:methods
C417561:Alexa 488 hydrazide; D002147:Calmodulin; D006834:Hydrazines; D010455:Peptides; D011994:Recombinant Proteins; D024461:Myosin Type I