Abstract
Recoverin (Rec) is a prototypical calcium sensor protein primarily expressed in the vertebrate retina. The binding of two Ca2+ ions to the functional EF-hand motifs induces the extrusion of a myristoyl group that increases the affinity of Rec for the membrane and leads to the formation of a complex with rhodopsin kinase (GRK1). Here, unbiased all-atom molecular dynamics simulations were performed to monitor the spontaneous insertion of the myristoyl group into a model multicomponent biological membrane for both isolated Rec and for its complex with a peptide from the GRK1 target. It was found that the functional membrane anchoring of the myristoyl group is triggered by persistent electrostatic protein-membrane interactions. In particular, salt bridges between Arg43, Arg46 and polar heads of phosphatidylserine lipids are necessary to enhance the myristoyl hydrophobic packing in the Rec-GRK1 assembly. The long-distance communication between Ca2+-binding EF-hands and residues at the interface with GRK1 is significantly influenced by the presence of the membrane, which leads to dramatic changes in the connectivity of amino acids mediating the highest number of persistent interactions (hubs). In conclusion, specific membrane composition and allosteric interactions are both necessary for the correct assembly and dynamics of functional Rec-GRK1 complex.
Highlights
The small (23 kDa) Ca2+-binding protein recoverin (Rec) is a prototypical member of the neuronal calcium sensor (NCS) family mostly expressed in vertebrate photoreceptors, where it plays a role in the regulation of the phototransduction cascade [1,2]
Seven 200 ns m. Tbhreaneextwenassivseimalul-laattoemd abnydmcooalersceu-lgarraidnyednamics (MD) replicas were run for each different simulated state (Rec-R and Rec-GRK1), two of which started from the protein at 1 nm distance from the membrane bilayer
A number of experimental studies based on Nuclear Magnetic Resonance (NMR) [5,28,38,45], surface plasmon resonance [7,12,46,47] and dynamic light scattering [48,49] all confirmed that the binding of two Ca2+ ions to the functional motifs EF2 and EF3 in Rec triggers the myristoyl switch process, which by extruding the acyl chain from the protein milieu leads to the significant exposure of a hydrophobic crevice eventually necessary to accommodate the GRK1 target
Summary
The small (23 kDa) Ca2+-binding protein recoverin (Rec) is a prototypical member of the neuronal calcium sensor (NCS) family mostly expressed in vertebrate photoreceptors, where it plays a role in the regulation of the phototransduction cascade [1,2]. Light triggers a transient drop in the intracellular concentration of Ca2+ and such change is promptly detected by a group of calcium sensors including Rec, which reversibly bind to specific targets in a Ca2+-dependent manner. This specific binding permits the complex regulation of the signaling cascade and is fundamental to bring the cell back to the dark state, where light sensitivity is maximal [3]. The peculiar physicochemical details have been long investigated [6,12,13]
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