Abstract
Purpose :
RD3 regulates retinal membrane guanylyl cyclase (RetGC) through blocking its activation by calcium sensor proteins, GCAPs, while promoting RetGC accumulation in photoreceptor outer segment. Mutations in RD3 cause severe recessive photoreceptor degeneration in human LCA12 patients and rd3 mice. The purpose of this study was to establish the three-dimensional structure of a human RD3.
Methods :
Soluble forms of RD3 were produced by site-directed mutagenesis and the resultant RD3 variants were tested for RetGC1 activation in vitro, binding to RetGC1 in HEK293 cells, and analyzed by size-exclusion chromatography and multi-angle light scattering. Ultimately, the solution structure of a soluble RD3 was established using NMR spectroscopy and restrained molecular dynamics simulations.
Results :
High propensity of RD3 to aggregate in solution, which previously prevented its structural analysis, was overcome by producing a soluble variant of human RD3 (RD3d), that was monomeric and retained the ability to bind and regulate RetGC. The NMR solution structure of RD3d revealed an elongated backbone structure (70Å long, 30Å wide), consisting of a four-helix bundle with a long unstructured loop between helices 1 and 2. RD3 residues implicated in RetGC binding based on mutational analysis mapped to a localized contiguous area on the structure involving a loop between helices 2 and 3 and adjacent parts of helices 3 and 4. The NMR structure of RD3 was additionally validated, by introducing larger hydrophobic residues in positions 29 and 85 disrupting packing in the hydrophobic core and lowering the RD3 apparent affinity for RetGC1. Conversely, introducing Val in position 93 stabilized the hydrophobic core and increased the RD3 affinity for the cyclase.
Conclusions :
The three-dimensional structure of RD3 has been determined by NMR and now provides a structural basis for further elucidating mechanisms of RD3 interaction with RetGC and their abnormalities leading to retinal diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.