Supramolecular complexes of GCAP1: towards the development of effective biologics for inherited retinal dystrophies

Guanylate Cyclase Activating Protein 1 (GCAP1) is a neuronal Ca ²⁺ -sensor protein expressed in photoreceptors where it regulates the enzymatic activity of retinal Guanylate Cyclase 1 (GC1) in a Ca ²⁺ -dependent manner. Recently, over 20 missense mutations in GUCA1A (encoding for GCAP1) have been associated with inherited autosomal dominant retinal diseases, namely cone dystrophy (COD) and cone-rod dystrophy (CORD). Since GCAP1 is known to be a functional dimer, COD/CORD patients present a heterogeneous pool of GCAP1 assemblies constituted by wild-type and mutated homodimers and heterodimers. Here, we present an integrated in silico and biochemical investigation on the effects of the E111V substitution, associated with a severe form of CORD, on GCAP1 homo- and hetero-dimerization. Despite inducing a constitutive activation of GC1 due to impaired Ca ²⁺ -binding in the high-affinity EF-hand 3 motif, the E111V substitution did not affect either the homo- or the hetero dimerization process as clearly highlighted by aSEC and molecular docking experiments. Indeed, both variants exhibited micromolar monomer-dimer equilibrium constants in the presence of both Mg ²⁺ and Ca ²⁺⁺ , indicating that at physiological cellular concentrations both variants are predominantly monomers under Ca ²⁺ -loaded and, to a lesser extent, Mg ²⁺ -loaded conditions. Molecular docking and dynamics simulations confirmed chromatographic results highlighting slight alterations in free energy of binding involving the pathogenic E111V variant in the Ca ²⁺ -bound state and increased mobility over time affecting the Ca ²⁺ -coordinating EF3 motif. In addition, to evaluate possible therapeutic approaches, the regulation of the catalytic activity of GC1 by WT and E111V-GCAP1 was studied in the presence of retinal degeneration protein 3 (RD3), an α-helical protein that strongly inhibits GC1, and a RD3-derived peptide (RD3ppt) which encompasses a region of RD3 that is essential for its inhibitory activity. GC1 activity assays in the presence of RD3ppt suggest that the enzymatic activity is partially inhibited by the peptide at low micromolar concentrations when GCAP1 variants are present. The incomplete shut down of GC1 by RD3 could be explained by the interaction occurring between RD3 and GCAP1, known to form a complex with GC1 in the endoplasmic reticulum. This fundamental interaction was here investigated spectroscopically and in silico , unveiling major structural rearrangements upon complex formation. Interestingly, the full RD3 protein was able to better modulate GC1 activity and restore the abnormal cGMP production induced by the pathogenic E111V-GCAP1 variant to a physiological level.