Structural profiling of conformational transitions in recoverin orthologs

The neuronal calcium sensor recoverin is essentially involved in the termination and recovery of the photoresponse by calcium-dependent inhibition of rhodopsin kinase (GRK1). The interaction with GRK1 is facilitated by a pronounced conformational change upon calcium binding, the calcium-myristoyl switch. In contrast to mammalian rod and cone photoreceptors which share the same recoverin isoform, the retina of the zebrafish, an emerging model organism for cone phototransduction, expresses four recoverin isoforms of which only one is expressed in rods. The abundance of cone zRec isoforms with high sequence identity raised questions regarding their physiological significance and individual functions. Recent studies suggested functional differences indicated by their individual subcellular localization and calcium-sensitivities. However, structural data explaining functional differences between zRec isoforms is lacking. We evaluated calcium-dependent changes in cysteine accessibility and intrinsic fluorescence of myristoylated and non-myristoylated recoverins and supplemented the experimental data with structural modelling approaches. These approaches were employed to provide a structural interpretation of the experimentally determined accessibilities and identify the individual cysteine residues most likely responsible for the experimental observations. The accessibility of the conserved residue C39 that is closely associated with the myristoyl-binding pocket showed only slight variability among myristoylated isoforms whereas non-myristoylated isoforms displayed considerable individual differences. Bicelles and GRK1 individually affected cysteine accessibility. Pronounced differences between zRec isoforms were observed in their intrinsic fluorescence spectra, pointing to different modes of calcium-dependent conformational changes between recoverin isoforms. The differences observed in intrinsic fluorescence and cysteine accessibilities indicate slight structural variations that could impact target regulation.