Structural and functional investigation of RD3-GCAP1 interaction in retinal photoreceptors under normal and disease conditions

Guanylate Cyclase Activating Protein 1 (GCAP1) and Retinal Degeneration Protein 3 (RD3) are key regulators of retinal guanylate cyclase 1 (GC1), whose dysregulation leads to inherited retinal dystrophies (IRDs). While GCAP1 mutations cause constitutive GC1 activation and photoreceptor degeneration, RD3 acts as a potent cyclase inhibitor essential for proper GC1 trafficking. Here, we investigated the molecular interaction between GCAP1 and RD3 as well as its perturbation by IRD-associated GCAP1 mutations (D100G, N104H, E111V, E155G) using NMR spectroscopy, surface plasmon resonance, AlphaFold3 modeling, enzymatic assays, and their localization via immunohistochemistry. The results demonstrate that the GCAP1-RD3 interaction is strongly Ca 2+ -dependent, with Ca 2+ -bound GCAP1 exhibiting micromolar affinity for RD3 (K D ~ 1.6 μM) and Mg 2+ -bound GCAP1 showing much weaker binding. Strikingly, the E111V mutation completely abolishes RD3 binding, whereas other variants retain interaction with differential kinetic properties. AlphaFold3 modeling, validated by NMR data, reveals that GCAP1 residues involved in RD3 binding overlap with those residues that mediate GCAP1 dimerization and GC1 interaction. Functional assays demonstrate that RD3 inhibits GC1 cyclase activity through dual mechanisms: direct binding to GC1 and GCAP1-mediated inhibition. Remarkably, RD3 rescues GC1 dysregulation caused by all tested GCAP1 mutations, regardless of their ability to interact with RD3. Immunohistochemistry reveals co-localization of GCAP1, RD3, and GC1 in photoreceptor inner segments and synaptic terminals, where Ca 2+ concentrations favor complex formation. Our findings suggest that the Ca 2+ gradient across the connecting cilium acts as a biochemical switch controlling RD3-GCAP1 interaction, and support RD3-based protein delivery as a mutation-independent therapeutic strategy for GCAP1-associated retinal dystrophies. • GCAP1-RD3 interaction is strongly Ca 2+ -dependent. • E111V-GCAP1 completely abolishes RD3 binding. • RD3 inhibits GC1 via dual direct and GCAP1-mediated mechanisms. • RD3 rescues GC1 dysregulation by all IRD-associated GCAP1 variants. • Ca 2+ gradient may act as biochemical gate for RD3-GCAP1 complex.