Binding-Interface Dynamics between Calmodulin and its Targets Revealed using Nonperturbative Infrared Probe Groups

Germany, Institute of Pharmacology and Toxicology, University of W€urzburg, W€urzburg, Germany. Phosphorylation is a ubiquitous post-translational modification that has been implicated in a myriad of biological functions but the underlying mechanism of action can be unclear. Here we study the phosphorylation-induced partial unfolding reaction in Raf Kinase Inhibitory Protein (RKIP), a dual function protein that regulates key pathophysiological states including heart disease and cancer. RKIP transitions between inhibition of Raf/MAPK to activation of Protein Kinase A via phosphorylation of a serine on RKIP. We show by NMR and X-ray crystallography that switching is due to a ’theft’ by the phosphoserine of a lysine involved in a salt bridge with a pair of carboxylic acids. The helical region containing the phosphorylation site remains intact whereas the region with the acidic groups unfolds, thereby switching RKIP’s preferred binding partner. A database search finds candidates that have the same structural motif underlying the theft mechanism. Three of them, Bax (1), troponin I & C (2), and Early endosome antigen 1 (3), had been more extensively characterized by mutations, and the results can be explained by a salt-bridge theft. These findings identify a facile and evolutionarily accessible mechanism for reorganizing a salt bridge network with only a single mutation to trigger a functional switch. We anticipate that the salt-bridge theft mechanism can be identified in other proteins and complexes. 1. Arokium, H., et al., (2007) J Biol Chem 282, 35104. 2. Kooij, V et al., (2013) PLoS One. 8, e74847. 3. Ramanathan, H. N., Zhang, G., and Ye, Y. (2013) Cell Biosci 3, 24.