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Functional Study and Mapping Sites for Interaction with the Target Enzyme in Retinal Degeneration 3 (RD3) Protein
Journal article   Open access   Peer reviewed

Functional Study and Mapping Sites for Interaction with the Target Enzyme in Retinal Degeneration 3 (RD3) Protein

Igor V Peshenko, Elena V Olshevskaya and Alexander M Dizhoor
The Journal of biological chemistry, v 291(37), pp 19713-19723
09 Sep 2016
DOI: https://doi.org/10.1074/jbc.M116.742288
PMID: 27471269
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1074/jbc.m116.742288View
Published, Version of Record (VoR)Open Access (License Unspecified),  Open
url
https://doi.org/10.1074/jbc.M116.742288View
Published, Version of Record (VoR) Open

Abstract

Amino Acid Substitution Animals Binding Sites Cell Line Guanylate Cyclase - genetics Guanylate Cyclase - metabolism HEK293 Cells Humans Mice Mice, Knockout Mutation, Missense Nuclear Proteins - genetics Nuclear Proteins - metabolism Protein Binding Protein Structure, Secondary Receptors, Cell Surface - genetics Receptors, Cell Surface - metabolism
Retinal degeneration 3 (RD3) protein, essential for normal expression of retinal membrane guanylyl cyclase (RetGC) in photoreceptor cells, blocks RetGC catalytic activity and stimulation by guanylyl cyclase-activating proteins (GCAPs). In a mouse retina, RD3 inhibited both RetGC1 and RetGC2 isozymes. Photoreceptors in the rd3/rd3 mouse retinas lacking functional RD3 degenerated more severely than in the retinas lacking both RetGC isozymes, consistent with a hypothesis that the inhibitory activity of RD3 has a functional role in photoreceptors. To map the potential target-binding site(s) on RD3, short evolutionary conserved regions of its primary structure were scrambled and the mutations were tested for the RD3 ability to inhibit RetGC1 and co-localize with the cyclase in co-transfected cells. Substitutions in 4 out of 22 tested regions, (87)KIHP(90), (93)CGPAI(97), (99)RFRQ(102), and (119)RSVL(122), reduced the RD3 apparent affinity for the cyclase 180-700-fold. Changes of amino acid sequences outside the Lys(87)-Leu(122) central portion of the molecule either failed to prevent RD3 binding to the cyclase or had a much smaller effect. Mutations in the (93)CGPAI(97) portion of a predicted central α-helix most drastically suppressed the inhibitory activity of RD3 and disrupted RD3 co-localization with RetGC1 in HEK293 cells. Different side chains replacing Cys(93) profoundly reduced RD3 affinity for the cyclase, irrespective of their relative helix propensities. We conclude that the main RetGC-binding interface on RD3 required for the negative regulation of the cyclase localizes to the Lys(87)-Leu(122) region.

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Web of Science research areas
Biochemistry & Molecular Biology
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