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G protein-coupled receptor kinase 1 (GRK1) or rhodopsin kinase is under specific control of the neuronal Ca 2+ -sensor protein recoverin, which is a critical feedback mechanism responsible for the modulation of the shape and sensitivity of the rod cell photoresponse. This process requires the precise matching of interacting protein surfaces and the dynamic changes in protein conformations. Here we study the molecular recognition process of recoverin and GRK1 by testing the hypothesis of a cation-π interaction pair in the recoverin-GRK1 complex. The critical role of residue K192 in recoverin was investigated by site-directed mutagenesis and subsequent structural and functional analysis. The following methods were used: isothermal titration calorimetry, fluorescence and circular dichroism spectroscopy, Ca 2+ -dependent membrane binding, and protein-protein interaction analysis by back scattering interferometry and surface plasmon resonance. While neutralizing the charge at K in the mutant K192L did not prevent binding of recoverin to GRK1, reversing the charge from K to E led to more distortions in the interaction process, but both mutations increased the stability of the protein conformation. Molecular dynamics simulations provided an explanation for these findings as they let us suggest that residue 192 per se is not a major stabilizer of the interaction between recoverin and its target but rather that the native K is involved in a network of switching electrostatic interactions in wild-type recoverin.

Recoverin (Rv), a small Ca(2+)-binding protein that inhibits rhodopsin kinase (RK), has four EF hands, two of which are functional (EF2 and EF3). Activation requires Ca(2+) in both EF hands, but crystal structures have never been observed with Ca(2+) ions in both sites; all previous structures have Ca(2+) bound to only EF3. We suspected that this was due to an intermolecular crystal contact between T80 and a surface glutamate (E153) that precluded coordination of a Ca(2+) ion in EF2. We constructed the E153A mutant, determined its X-ray crystal structure to 1.2 Å resolution, and showed that two Ca(2+) ions are bound, one in EF3 and one in EF2. Additionally, several other residues are shown to adopt conformations in the 2Ca(2+) structure not seen previously and not seen in a second structure of the E153A mutant containing Na(+) instead of Ca(2+) in the EF2 site. The side-chain rearrangements in these residues form a 28 Å allosteric cascade along the surface of the protein connecting the Ca(2+)-binding site of EF2 with the active-site pocket responsible for binding RK.