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. 2013 Jun;14(6):520-6.
doi: 10.1038/embor.2013.44. Epub 2013 Apr 12.

Insights into congenital stationary night blindness based on the structure of G90D rhodopsin

Ankita Singhal  1 , Martin K Ostermaier, Sergey A Vishnivetskiy, Valérie Panneels, Kristoff T Homan, John J G Tesmer, Dmitry Veprintsev, Xavier Deupi, Vsevolod V Gurevich, Gebhard F X Schertler, Joerg Standfuss
Affiliations

Insights into congenital stationary night blindness based on the structure of G90D rhodopsin

Ankita Singhal et al. EMBO Rep. 2013 Jun.

Abstract

We present active-state structures of the G protein-coupled receptor (GPCRs) rhodopsin carrying the disease-causing mutation G90D. Mutations of G90 cause either retinitis pigmentosa (RP) or congenital stationary night blindness (CSNB), a milder, non-progressive form of RP. Our analysis shows that the CSNB-causing G90D mutation introduces a salt bridge with K296. The mutant thus interferes with the E113Q-K296 activation switch and the covalent binding of the inverse agonist 11-cis-retinal, two interactions that are crucial for the deactivation of rhodopsin. Other mutations, including G90V causing RP, cannot promote similar interactions. We discuss our findings in context of a model in which CSNB is caused by constitutive activation of the visual signalling cascade.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Comparison of the G90D-GαCT2 and G90D structures with metarhodopsin-II [12]. All-trans (yellow) and cis-retinal isomers (orange), the site of retinal attachment K296 (blue slate), the retinal counterion E113 (blue slate), GαCT peptides (cyan) and the constitutively activating mutations G90D and M257Y (green) are shown as spheres. The inset shows a closer view of the side chains that contribute to the retinal-binding pocket (within 4 Å of the retinal+the counterion E113) shown as sticks. Formation of a salt bridge (red dotted lines) between G90D (green) and K296 together with electrostatic interference with the E113 disfavours covalent binding of retinal resulting in a mixed retinal population, illustrated here as 13-cis and 9–13-di-cis-retinal.
Figure 2
Figure 2
Impact of the G90D mutation on thermal stability and binding of arrestin. Thermal stability (A) of wild type (WT) and G90D opsin (all in the stabilizing N2C/D282C background) in presence and absence of retinal. Graphs indicate the mean melting temperature (Tm50)±s.d. of the melting temperatures obtained from four fluorescent thermal shift experiments. (B–D) Direct binding assay of radiolabeled arrestin-1 to different forms of rhodopsin (phosphorylated rhodopsin (R-P), phosphorylated opsin (P-Ops) and unphosphorylated rhodopsin (R)) in nanodiscs. Binding experiments of wild type (B), constitutively active M257Y (C) and G90D (D) rhodopsin were performed either in the dark or under room light (*). Means and s.d. were obtained from four experiments.
Figure 3
Figure 3
Electron density (2Fo-Fc contoured at 1.5 sigma, blue and Fo-Fc contoured at 3.5 sigma, green) of the G90D-GαCT retinal-binding pocket calculated after simulated annealing refinement with the G90D side chain omitted. The obtained map shows a clear difference peak for the introduced G90D mutation and the salt bridge (red dashes) with K296. Clear positive density close to the position of all-trans-retinal in metarhodopsin-II indicates that some retinal was retained in the binding pocket during crystallization. Formation of the G90D-K296 salt bridge interferes with formation of a covalent bond and results in density that is most compatible with a mixture of retinals. For comparison, the 9,13-di-cis retinal isomer is shown as orange sticks.
Figure 4
Figure 4
Comparison of congenital stationary night blindness (CSNB)- (green, lower left panel) and retinitis pigmentosa (RP)- (blue, lower right panel) causing mutations near the retinal-binding pocket of light-activated rhodopsin. All four CSNB mutations could be placed into the structure of active G90D rhodopsin in a favourable rotamer conformation and without introducing major clashes with the rest of the protein. Three CSNB mutations are able to form either van der Waals (blue dashes), or salt bridge interactions (red dashes) with K296, whereas A295V might alter the position of K296 more indirectly and interacts with the W265 activation switch. Nearby RP mutations do not show a similar pattern and point away from the retinal Schiff base (SB). The only exceptions are RP-causing mutations of K296, which cannot covalently bind retinal, and the uncharged G90V mutation that cannot form the same salt bridge towards K296 as the G90D mutation.
Figure 5
Figure 5
The molecular causes of congenital stationary night blindness. In healthy dim-light vision, (A) basal G protein transducin (Gt) activation by opsin and dark-state rhodopsin (red circles) is prevented by a salt bridge (dashed red line) between K296, the site of retinal attachment by means of a protonated Schiff base (SB) (black line) and the retinal counterion E113. Absorption of a photon leads to isomerization of retinal (to trans), proton transfer to E113, opening of the E113-K296 activation switch and a series of conformational changes in the seven-helix bundle (yellow circles). Subsequent phosphorylation by GRK1 increases affinity for arrestin-1, which quenches signalling by blocking the Gt-binding site. (B) The G90D mutation causes congenital stationary night blindness (CSNB) through constant background activation that desensitizes the visual system. Three not mutually exclusive mechanisms might cause this phenotype: (1) constant simulation by constitutively active opsin, (2) spontaneous activation by thermal isomerization of retinal or (3) a preactivated rhodopsin dark state. The G90D mutation introduces an extra charge that interferes (|_|) with the deactivating E113-K296 salt bridge [2, 17] and instead forms a salt bridge with K296 in the active opsin state. This salt bridge interferes with SB formation and prevents efficient opsin deactivation through covalent binding of 11-cis-retinal. Interference of G90D with stability of the retinal SB increases the rate of thermal isomerization and the basal activity of the receptor dark state.
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