Calcium binding to calmodulin leads to an N-terminal shift in its binding site on the ryanodine Receptor.
The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614-3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.
Rodney
G G
GG
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
Moore
C P
CP
Williams
B Y
BY
Zhang
J Z
JZ
Krol
J
J
Pedersen
S E
SE
Hamilton
S L
SL
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D005453:Fluorescence; D008969:Molecular Sequence Data; D011485:Protein Binding; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry
D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D014364:Tryptophan; D002118:Calcium