QUANTITATIVE ENDOPROTEINASE GLUC FOOTPRINTING OF COOPERATIVE CA2- PROTEOLYTIC SUSCEPTIBILITY OF E31 AND E87 INDICATES INTERDOMAIN INTERACTIONS( BINDING TO CALMODULIN )

Calmodulin is the primary eukaryotic intracellular calcium receptor. C ooperative calcium binding to two sites in each of two domains drives large conformational changes that enable it to activate target protein s. An understanding of the molecular mechanism of cooperativity requir es 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 mon itor residue-specific conformational changes within calmodulin as calc ium binds, we have developed a new quantitative proteolytic footprinti ng method using endoproteinase GluC (EndoGluC). Under conditions of ve ry 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 indivi dual peptide bonds. Quantitative susceptibility profiles were resolved for two positions: E31-L32, in site I in the N-terminal domain, and E 87-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. Susc eptibility was induced by calcium binding to sites III and IV, followe d by protection induced by calcium binding to sites I and II. This ind icated that calmodulin must adopt at least three distinguishable confo rmations and suggested that the two domains interact. Model-dependent equilibrium constants were resolved from the EndoGluC susceptibility p rofiles for E31 and E87; they indicated cooperative binding within eac h domain. Approaches taken to validate this proteolytic footprinting m ethod are described.