Label-free quantification of calcium-sensor targeting to photoreceptor guanylate cyclase and rhodopsin kinase by backscattering interferometry

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.

Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction

“…In comparison, K D values determined for GCAP-2 and the human, full-length guanylyl cyclase (418 ± 135 nM) via backscattering interferometry, are consistent with the results shown herein (Sulmann et al, 2017 ). However, no interaction between GCAP-2 and the isolated domain of human ROS-GC-1 residues 496-806 (comprising the juxtamembrane and kinase homology domains) could be demonstrated in those experiments.…”

“…The 657 WTAPELL 663 motif in the C -terminal part of the KHD of ROS-GC1 engages in signal transfer processes to activate the catalytic domain, but not in the binding reaction to GCAPs (Duda et al, 2011 ). The hypothesis of separate interaction sites for GCAP-1 and GCAP-2 at ROS-GC1 is supported by affinity determinations via backscattering interferometry using different constructs of the intracellular domain (Sulmann et al, 2017 ).…”

Photoreceptor Guanylate Cyclase (GUCY2D) Mutations Cause Retinal Dystrophies by Severe Malfunction of Ca2+-Dependent Cyclic GMP Synthesis

“…These mutations may change the overall structure of the protein preventing activation of GC-E by GCAPs. Direct binding of GCAP1 could be impaired, since either its binding site or an important activity control site is located in this domain ( Sulmann et al, 2017 ). Further, the KHD harbors a putative Mg 2+ binding site that is part of the nucleotide (ATP) binding site and is suggested to stabilize the active conformation of the catalytic domain by multiple hydrogen bonds ( Bereta et al, 2010 ).…”

CaF2nanoparticles as surface carriers of GCAP1, a calcium sensor protein involved in retinal dystrophies

“…Binding stoichiometry of GCAP1 interacting with NP was tested by isothermal titration calorimetry (ITC) as described earlier for other binding processes involving NCS proteins. 18,29,30 Briefly, ITC was performed using a VP-ITC instrument from MicroCal (Northampton, MA, USA) at T = 25°C. Purified GCAP1 or the mutant D100E was present in the recording cell in a decalcified buffer composed of 5 mM Tris-HCl pH 7.5 and 150 mM KCl.…”

“…Purified GCAP1 or D100E was added to washed HEK cell membranes at a fixed saturating concentration of 10 µM and GC activity was measured in the 20 nM-100 µM range using a Ca 2+ /EGTA buffer system allowing to determine the IC 50 value. [25][26][27][28][29] Secondly, we incubated GC in HEK cell membranes with increasing concentrations of WT or D100E GCAP1 (0-20 µM) under conditions, in which GCAPs were Mg 2+ -bound (Ca 2+ -free) (incubation time 5 min). Both activation series were run in the absence and presence of CaF 2 NP.…”