Quantitative endoproteinase GluC footprinting of cooperative Ca2+ binding to calmodulin: proteolytic susceptibility of E31 and E87 indicates interdomain interactions.
Calmodulin is the primary eukaryotic intracellular calcium receptor. Cooperative calcium binding to two sites in each of two domains drives large conformational changes that enable it to activate target proteins. An understanding of the molecular mechanism of cooperativity requires determination of the conformational states populated by calmodulin, the intrinsic free energies of binding calcium to four sites, and the nature and degree of intradomain and interdomain interactions. To monitor residue-specific conformational changes within calmodulin as calcium binds, we have developed a new quantitative proteolytic footprinting method using endoproteinase GluC (EndoGluC). Under conditions of very limited proteolysis, cleavage occurred at only five of the sixteen positions possible in calmodulin. The relative abundance of fragments indicated that calcium induced changes in the susceptibility of individual peptide bonds. Quantitative susceptibility profiles were resolved for two positions: E31-L32, in site I in the N-terminal domain, and E87-A88, preceding site III in the C-terminal domain. In apocalmodulin, E87-A88 was susceptible to EndoGluC; calcium binding to sites III and IV caused monotonic protection from proteolysis. The response of E31-L32 was biphasic. In apocalmodulin, it was resistant to cleavage. Susceptibility was induced by calcium binding to sites III and IV, followed by protection induced by calcium binding to sites I and II. This indicated that calmodulin must adopt at least three distinguishable conformations and suggested that the two domains interact. Model-dependent equilibrium constants were resolved from the EndoGluC susceptibility profiles for E31 and E87; they indicated cooperative binding within each domain. Approaches taken to validate this proteolytic footprinting method are described.
Pedigo
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Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1109.
Shea
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MA
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000737:chemistry
D002147:Calmodulin; D011994:Recombinant Proteins; D014443:Tyrosine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002118:Calcium