Retinal degeneration 3 (RD3) protein, a retinal guanylyl cyclase regulator, forms a monomeric and elongated four-helix bundle

Abstract

Retinal degeneration 3 (RD3) protein promotes accumulation of retinal membrane guanylyl cyclase (RetGC) in the photoreceptor outer segment and suppresses RetGC activation by guanylyl cyclase-activating proteins (GCAPs). Mutations truncating RD3 cause severe congenital blindness by preventing the inhibitory binding of RD3 to the cyclase. The high propensity of RD3 to aggregate in solution has prevented structural analysis. Here, we produced a highly soluble variant of human RD3 (residues 18 -160) that is monomeric and can still bind and negatively regulate RetGC. The NMR solution structure of RD3 revealed an elongated backbone structure (70 Å long and 30 Å wide) consisting of a four-helix bundle with a long unstructured loop between helices 1 and 2. The structure reveals that RD3 residues previously implicated in the RetGC binding map to a localized and 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 validated by mutagenesis. Introducing Trp 85 or Phe 29 to replace Cys or Leu, respectively, disrupts packing in the hydrophobic core and lowers RD3's apparent affinity for RetGC1. Introducing a positive charge at the interface (Glu 32 to Lys) also lowered the affinity. Conversely, introducing Val in place of Cys 93 stabilized the hydrophobic core and increased the RD3 affinity for the cyclase. The NMR structure of RD3 presented here provides a structural basis for elucidating RD3-RetGC interactions relevant for normal vision or blindness.

Retinal guanylyl cyclase activation by calcium sensor proteins mediates photoreceptor degeneration in an rd3 mouse model of congenital human blindness

“…Currently little is known about the mechanisms of RD3 interaction with the cyclase. The structures of GCAPs (30 -32) and recently RD3 (33) have been established; however, the structure of the cyclase itself still presents a major barrier for understanding the molecular dynamics of the complex. To date, the threedimensional structure of RetGC has been resolved only for its catalytic core (34,35), not for those domains that present most important regulatory parts.…”

“…RD3 and GCAP (11,17) not only have highly dissimilar protein sequences (Ͻ5% overall identity and Ͻ9% similarity in pairwise alignment; European Bioinformatics Institute EMBOS Needle on-line tool) but also drastically dissimilar three-dimensional structures. The guanylyl cyclasebinding interface on RD3 (22,33) occupies the central part of RD3 elongated bundle of ␣-helices, whereas the cyclase-binding interface of GCAP occupies portions of three helix-loophelix EF-hand domains (38). This suggests that most likely the binding sites for RD3 and GCAP on RetGC are also nonidentical.…”

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

“…The molecular structure of RetGC remains largely unknown and mutational analysis of the cyclase presents a major challenge because of the larger size of the enzyme and the complexity of its regulation. Another major challenge presents high propensity of RD3 to precipitate at concentrations required for structural analyses ( 24 , 27 ). However, the three-dimensional structure of RD3 core, an elongated bundle of four α-helices ( Fig.…”

“…However, the three-dimensional structure of RD3 core, an elongated bundle of four α-helices ( Fig. 1 ), was recently established using a soluble variant of RD3 that retained attenuated affinity for the cyclase ( 27 ). The preliminary testing of several fragments in RD3 primary structure that contained surface-exposed and buried in the core structure residues indicated that the cyclase-binding interface on RD3 includes the central portion of the helical bundle ( Fig.…”

“…The preliminary testing of several fragments in RD3 primary structure that contained surface-exposed and buried in the core structure residues indicated that the cyclase-binding interface on RD3 includes the central portion of the helical bundle ( Fig. 1 ) and that the part of the bundle forming the functional interface with the cyclase involves helices 3 and 4 ( 24 , 27 ). Nonetheless, the identities of the residues on the surface of RD3 that are essential for its functional contact with the cyclase remained unclear, in part because the full-size RD3 remains unsuitable for structural analyses.…”