Abstract
Quantification of protein binding to membrane proteins is challenging and a limited set of methods is available to study such systems. Here we employed backscattering interferometry (BSI), a free-solution label-free method with high sensitivity, to quantify the interaction of neuronal Ca2+-Sensor proteins with their targets operating in phototransduction. We tested direct binding of guanylate cyclase–activating proteins (GCAP1 and GCAP2) to their membrane target guanylate cyclase 1. The regulatory mechanism of GCAPs including their binding interface in the target is unresolved. Here we used a label-free, free-solution assay method based on BSI to determine binding constants of GCAP1 and GCAP2 to the full-length membrane-bound guanylate cyclase type 1. GCAP1 and GCAP2 bound to different regions on the target guanylate cyclase with submicromolar affinity (apparent KD-values of 663 ± 121 nM and 231 ± 63 nM for Ca2+-free GCAP1 and GCAP2, respectively). A guanylate cyclase construct containing the juxta-membrane and kinase homology domain harbored an exclusive binding site for GCAP1 with similar affinities as the full-length protein, whereas GCAP2 did not bind to this region. We provide a model in which GCAP1 and GCAP2 do not share a single binding site to the target, thus cannot exchange upon fluctuating Ca2+ levels.
Highlights
Retinal diseases like Lebers congenital amourosis (LCA) and Cone-Rod-Dystrophies (CORD) correlate with mutations in the GUCY2D gene coding for photoreceptor guanylate cyclases (GCs)-E12; (iii) mutations in the GUCA1A gene coding for GCAP1 correlate with cone, cone-rod and macular dystrophies in patients suffering from visual dysfunction, which is likely caused by an impaired operation of the GC-E/ GCAP1 complex, since GC-E is the preferred target of GCAP113
In the present study we investigated GC-E/GCAP interactions using a novel technique called Backscattering Interferometry (BSI), which allows label- and immobilization-free interaction analysis in physiologically relevant matrices at high sensitivity and in small volumes[20,21,22]
Like GCAP1 and 2, it belongs to the family of neuronal Ca2+-sensor (NCS) proteins and it exhibits similar three-dimensional folding[5,7]
Summary
Retinal diseases like Lebers congenital amourosis (LCA) and Cone-Rod-Dystrophies (CORD) correlate with mutations in the GUCY2D gene coding for photoreceptor GC-E12; (iii) mutations in the GUCA1A gene coding for GCAP1 correlate with cone, cone-rod and macular dystrophies in patients suffering from visual dysfunction, which is likely caused by an impaired operation of the GC-E/ GCAP1 complex, since GC-E is the preferred target of GCAP113. Deciphering the control mechanisms of cGMP synthesis in the GC-E/GCAP1 complex is important in understanding cGMP homeostasis in photoreceptor cells in health and disease. It was possible to test both of the currently proposed binding models: one, in which GCAP1 and GCAP2 interact at different sites in GC-E, and the other one where both NCS proteins share a single binding site. Control BSI assays were performed with the photoreceptor NCS protein recoverin that is known to bind to rhodopsin kinase GRK1 in a Ca2+-dependent manner[23,24,25,26,27], enabling the determination of specific affinity in this complex physiological system
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