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Two clusters of surface-exposed amino acid residues enable high-affinity binding of retinal degeneration-3 (RD3) protein to retinal guanylyl cyclase
Journal article   Open access   Peer reviewed

Two clusters of surface-exposed amino acid residues enable high-affinity binding of retinal degeneration-3 (RD3) protein to retinal guanylyl cyclase

Igor V Peshenko and Alexander M Dizhoor
The Journal of biological chemistry, v 295(31), pp 10781-10793
31 Jul 2020
DOI: https://doi.org/10.1074/jbc.RA120.013789
PMID: 32493772
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1074/jbc.ra120.013789View
Published, Version of Record (VoR)Open Access (License Unspecified),  Open
url
https://doi.org/10.1074/jbc.RA120.013789View
Published, Version of Record (VoR) Open

Abstract

Amino Acid Substitution Animals Cattle Eye Proteins - genetics Eye Proteins - metabolism Guanylate Cyclase - genetics Guanylate Cyclase - metabolism Guanylate Cyclase-Activating Proteins - genetics Guanylate Cyclase-Activating Proteins - metabolism HEK293 Cells Humans Mutation, Missense Protein Binding Receptors, Cell Surface - genetics Receptors, Cell Surface - metabolism
Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase-activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC. We tested the effects of these substitutions and deletions by reconstituting purified RD3 variants with GCAP1-activated human RetGC1. Although the vast majority of the surface-exposed residues tolerated substitutions without loss of RD3's inhibitory activity, substitutions in two distinct narrow clusters located on the opposite sides of the molecule effectively suppressed RD3 binding to the cyclase. The first surface-exposed cluster included residues adjacent to Leu in the loop connecting helices 1 and 2. The second cluster surrounded Arg on a surface of helix 3. Single substitutions in those two clusters drastically, up to 245-fold, reduced the IC for the cyclase inhibition. Inactivation of the two binding sites completely disabled binding of RD3 to RetGC1 in living HEK293 cells. In contrast, deletion of 49 C-terminal residues did not affect the apparent affinity of RD3 for RetGC. Our findings identify the functional interface on RD3 required for its inhibitory binding to RetGC, a process essential for protecting photoreceptors from degeneration.

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Web of Science research areas
Biochemistry & Molecular Biology
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