Defining protein-protein interactions using site-directed spin-labeling: the binding of protein kinase C substrates to calmodulin.

EPR spectroscopy was used to examine protein-protein interactions between calmodulin and spin-labeled peptides based on the protein kinase C substrate domains of the myristoylated alanine rich C kinase substrate (MARCKS) and neuromodulin. When bound to calmodulin, the C- and N-terminal ends of a 25 residue MARCKS derived peptide exhibited large amplitude motion on the nanosecond time scale and were accessible to paramagnetic agents in aqueous solution. However, residues 5-23 were highly protected and in contact with side chains from calmodulin. These data are consistent with an alpha-helical configuration for this segment of MARCKS and with structures that have been obtained for other calmodulin-substrate complexes. For the 17 residue neuromodulin derived peptide, which is Ca2+ independent in its binding to calmodulin, oxygen collision rates demonstrate that one helical face of this peptide interacts strongly with calmodulin. The data are consistent with an interaction of this face specifically with the C-terminal lobe of calmodulin, where this lobe is either in an "open" or "semiopen" configuration. The EPR data also indicate that the N-terminal lobe of calmodulin is in contact with the peptide, but that this lobe is not as strongly associated with the peptide target. Overall, the binding pocket for neuromodulin appears to be less compact and more dynamic than that formed by MARCKS. This behavior has not previously been seen for calmodulin substrates, and it may play a role in the Ca2+ independent binding of this class of substrates. This work demonstrates the utility of EPR spectroscopy to define protein-protein interactions; in addition, oxygen collision frequencies obtained at buried sites appear to provide information on the conformational dynamics of proteins.