The kinase homology and dimerization domains in retinal membrane guanylyl cyclase 1 form the binding interface for the calcium sensor GCAP1.

Retinal membrane guanylyl cyclase (RetGC) regulated by guanylyl cyclase activating proteins (GCAPs) enables photoreceptor light sensitivity by producing cGMP to open cyclic nucleotide gated channels. The quaternary structure of the RetGC:GCAP complex remains unresolved. In this study, we conducted site-directed mutagenesis of RetGC1 based on the RetGC1:GCAP1 complex structure generated using AlphaFold 3 in order to identify the interface for GCAP1 on RetGC1. The structure of RetGC12:GCAP12 tetrameric complex from multiple independent trials of AlphaFold 3 modeling consistently positioned GCAP1 in contact with the kinase homology domain (KHD) and the dimerization domain (DD) of RetGC1. Residues in KHD and DD domains predicted to form direct contacts with GCAP1 were substituted to either disrupt or enhance GCAP1 binding by RetGC1 in vitro. Three residues in KHD predicted to be a part of the interface structure were most critical for binding GCAP1 based on biochemical analyzes - Asp639, Lys637, and Met641. Several predicted interface residues on DD domain also affected RetGC1 apparent affinity for GCAP1 - Ser819, Met823, Asp832, Glu833, and Glu837. The biochemical evidence supports the AlphaFold 3 structural model of the RetGC12:GCAP12 tetramer, in which the N-proximal semi-globule of each GCAP1 molecule binds KHD domain on one RetGC1 subunit and DD domains on both RetGC1 subunits of the RetGC1 homodimer, indicating that each GCAP1 molecule can directly regulate the activity of RetGC1 by altering the conformation of the cyclase homodimer.