VARIABLE CONFORMATION AND DYNAMICS OF CALMODULIN COMPLEXED WITH PEPTIDES DERIVED FROM THE AUTOINHIBITORY DOMAINS OF TARGET PROTEINS

Calcium-saturated calmodulin (CaM) can bind and activate many target p roteins through the direct association with the respective autoinhibit ory domains. The CaM binding sequences within the autoinhibitory domai ns of these proteins have little sequence homology, and the mechanisms associated with CaM's ability to recognize and productively bind with these variable sequences is unclear. Common structural features of Ca M bound,to five peptides that are homologous to the autoinhibitory dom ains of smooth muscle myosin light chain kinase, CaM-dependent protein kinase II alpha, the plasma membrane Ca-ATPase, a MARCKS homolog, and glycogen phosphorylase kinase were assessed using frequency-domain fl uorescence spectroscopy, In addition, the structural features of CaM c omplexed with the peptide melittin was also considered. We observe sim ilar decreases in the average fluorescence lifetime and similar increa ses in the solvent accessibility of N-(1-pyrenyl)maleimide (PM) bound at Cys(27) in calcium binding loop I in the amino terminal domain of C aM upon association with all six target peptides, Likewise, using fluo rescence resonance energy transfer to measure the spatial separation b etween the opposing globular domains in CaM, we observe a similar spat ial separation between the opposing globular domains of CaM bound to a ll six peptides. This indicates that CaM undergoes comparable structur al changes upon association with all six target peptides. However, the re are significant differences in the observed lifetime, solvent acces sibility, correlation time associated with the segmental rotational mo tion of PM-CaM, and in the spatial separation between the opposing glo bular domains in CaM upon association with the individual target pepti des, which indicates that CaM adopts a different tertiary structure th at is dependent on the structural features of the bound target peptide . The correlation times associated with the overall hydrodynamic prope rties of CaM complexed with all six peptides are nearly identical (phi (2) approximate to 10.6 +/- 0.4 ns) and are consistent with the known dimensions of CaM complexed to a peptide homologous to the CaM binding sequence of CaM-dependent protein kinase II alpha. Therefore, while t hese results are consistent with a common binding mechanism between Ca M and all six target peptides, they indicate that the binding domains of CaM adopt different tertiary structures that allow them to bind wit h the variable sequences found in the autoinhibitory domains of target proteins with high affinity.