Molecular Recognition of Rhodopsin Kinase GRK1 and Recoverin Is Tuned by Switching Intra- and Intermolecular Electrostatic Interactions

Abstract

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.

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling

“…Generally, residues of the dNCS-1 interface did not interfere with the GRK1-binding site [54], as far as could be predicted in the absence of a Ca 2+ -bound dimer structure. In any case, the increased structural plasticity suggested for dNCS-1 could have enhanced the stability of its complex with GRK1 and could be part of the conformational selection process [67,68], which seems relevant for the interactions of multifunctional NCS-1. An additional factor could be seen from the SPR studies, revealing 10-fold faster kinetics of GRK1 in association with dNCS-1, as compared to NCS-1 (see Table 2).…”

Regulation of retinal membrane guanylyl cyclase (RetGC) by negative calcium feedback and RD3 protein

“…A possible function of recoverin is to help rhodopsin, by extending the life of its active state, to more effectively propagate activation of the phototransduction cascade in dim light, before the receptor is quenched by phosphorylation and arrestin binding [ 1 , 7 , 16 , 47 , 48 ]. GRK1 is considered to be the primary target for Ca 2+ recoverin [ 1 , 129 ] although a more direct effect on PDE6 activity was also recently proposed [ 75 ] (Fig. 1 ).…”

Bringing the Ca 2+ sensitivity of myristoylated recoverin into the physiological range

“…Nonmyristoylated Rec was expressed and purified with the same procedure as for mRec, with the only difference being the absence of myristic acid and kanamycin during bacterial growth. The peptide encompassing the first 25 amino acids of GRK1 (MDFGSLETVVANSAFIAARGSFDAS [10], hereafter 'GRK1 peptide'), added with a neutral electric charge C-terminal tag (DGKGDK) for increased solubility [43] was purchased from GenScript, with a 97.1% purity, as assessed by HPLC. Peptide mass was estimated by amino acid analysis following acid hydrolysis.…”