Supramolecular complexes of GCAP1: implications for inherited retinal dystrophies

Abstract

Guanylate Cyclase Activating Protein 1 (GCAP1) is a calcium sensor that regulates the enzymatic activity of retinal Guanylate Cyclase 1 (GC1) in photoreceptors in a Ca²⁺/Mg²⁺ dependent manner. While point mutations in GCAP1 have been associated with inherited retinal dystrophies (IRDs), their impact on protein dimerization or on the possible interaction with the potent GC1 inhibitor RD3 (retinal degeneration protein 3) has never been investigated. Here, we integrate exhaustive in silico investigations with biochemical assays to evaluate the effects of the p.(E111V) substitution, associated with a severe form of IRD, on GCAP1 homo- and hetero-dimerization, and demonstrate that wild type (WT) GCAP1 directly interacts with RD3. Although inducing constitutive activation in GC1, the E111V substitution only slightly affects the dimerization of GCAP1. Both WT- and E111V-GCAP1 are predominantly monomeric in the absence of the GC1 target, however E111V-GCAP1 shows a stronger tendency to be monomeric in the Ca²⁺-bound form, corresponding to GC1 inhibiting state. Reconstitution experiments performed in the co-presence of WT-GCAP1, E111V-GCAP1 and RD3 restored nearly physiological regulation of the GC1 enzymatic activity in terms of cGMP synthesis and Ca²⁺-sensitivity, suggesting new scenarios for biologics-mediated treatment of GCAP1-associated IRDs.

Keywords: Biologics; Guanylate cyclase; RD3.