Structural role of the T94I rhodopsin mutation in congenital stationary night blindness

Abstract

Congenital stationary night blindness (CSNB) is an inherited and non-progressive retinal dysfunction. Here, we present the crystal structure of CSNB-causing T94I^2.61 rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin-II state by establishing a direct van der Waals contact with K296^7.43, the site of retinal attachment. This is in stark contrast to the light-activated state of the CSNB-causing G90D^2.57 mutation, where the charged mutation forms a salt bridge with K296^7.43 To find the common denominator between these two functional modifications, we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D^2.57 and the hydrophobic T94I^2.61 mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E113^3.28 We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients.

Keywords: G protein‐coupled receptors; congenital stationary night blindness; constitutive activity; rhodopsin; visual system.

Figure 1. Comparison of constitutively active rhodopsin mutants M257Y^6.40 (left 21), T94I^2.61 (middle), and G90D^2.57 (right 16)

All‐trans (yellow) and cis retinal isomers (orange); the side of retinal attachment K296^7.43 (blue slate); the retinal counterion E113^3.28 (blue slate); GαCT peptides (cyan); and the constitutively activating mutations M257Y^6.40, T94I^2.61, and G90D^2.57 (green) are shown as spheres. Palmitoylation at C323^H8 and glycosylation at N15^NT are shown as sticks. The lower panels compare the M257Y^6.40, T94I^2.61, and G90D^2.57 retinal binding pockets. All constructs also contain an additional thermostabilizing disulfide bridge, depicted as (c‐c), to enhance the stability 18 and crystallizability 19 without changing the rhodopsin activation pathway 20.

Figure EV1. Simulated annealing OMIT maps show the mutated position in the retinal binding pocket

Electron density maps (2F_o–F_c contoured at 1.8 sigma, blue and F_o–F_c contoured at 3.5 sigma, green) of the retinal binding pocket calculated after simulated annealing refinement with the side chain of G90D^2.57 and retinal (left) or T94I^2.61 (right) omitted. A clear difference peak indicates the introduced mutations in the vicinity of the retinal attachment site K296^7.43. The structure of G90D^2.57 resembles an active opsin state with a bound mixture of retinal isomers, while T94I^2.61 resembles the active metarhodopsin‐II intermediate with intact Schiff base and retinal in all‐trans conformation.

Figure EV2. Effect of CSNB mutants G90D^2.57 and T94I^2.61 on the thermal stability of the protein

The mean melting temperature Tm₅₀ was measured for WT, G90D^2.57, and T94I^2.61 in the presence or absence of retinal isomers in a fluorescence‐based thermal stability assay. Average melting temperature ± standard deviation (SD) was calculated over four independent measurements (n = 4).

Figure 2. Kinetic characterization of the rhodopsin dark state

1. A–C

   Thermal decay of dark state rhodopsin: WT (A), T94I (B), and G90D (C) rhodopsin. The spectra are normalized to the OD₂₈₀ and OD₅₀₀/OD₄₈₀ at t = 0.

2. D

   Absorption maximum plotted as a function of time and fitted to a single exponential decay.

3. E–G

   Retinal isomerization in the rhodopsin dark state: WT (E), T94I (F), and G90D (G) rhodopsin.

4. H

   The fraction of 11‐cis retinal is plotted as a function of time and fitted to a single exponential decay.

5. I–K

   SB stability in dark state rhodopsin: WT (I), T94I (J), and G90D (K) rhodopsin.

6. L

   OD₄₄₀ is plotted as a function of time and fitted to a single exponential decay.

Data information: All decay rates were measured at 55°C.

Figure 3. Accelerated molecular dynamic simulation of the rhodopsin dark state: WT, G90D^2.57, T94I^2.61, and T94I^2.61 with protonated E113^3.28

In the absence of the G90D^2.57 and T94I^2.61 dark state structure, both aspartate and isoleucine were placed in the WT (1GZM 48) using a favorable rotamer without introducing major clashes with the rest of the protein. However, both isoleucine and aspartate in the dark state structures introduced clashes with several amino acids and were therefore optimized using energy minimization before simulation. WT simulation was done with the protonated SB while in the G90D^2.57 system, both E113^3.28 and SB were protonated 36. The protonated state of E113^3.28 was unknown in T94I^2.61; therefore, simulation was done for both deprotonated and protonated E113^3.28. The T94I^2.61 dark state conformation with deprotonated E113^3.28 was similar to that of the WT and was stable, whereas the dark state of G90D^2.57 and T94I^2.61 with protonated E113^3.28 was unstable with a concomitant movement of E113^3.28 residue away from the SB, thereby causing movement of TM3 and opening of the E113^3.28‐SB activation switch. In contrast to the crystal structures, the orientation of the retinal β‐ionone ring in the simulations was variable with respect to the polyene chain. This indicates a degree of positional freedom within the hydrophobic binding pocket.

Figure 4. The molecular cause of congenital stationary night blindness (CSNB)

Disk membranes (upper panels) in the rod cells of healthy individuals (left) and CSNB patients (right) contain a normal distribution of rhodopsin. Interference of the T94I^2.61 and G90D^2.57 mutations with the E113^3.28‐SB activation switch (middle panels) leads to partial activation of a small portion of rhodopsins (orange). The resulting basal stimulation of the visual system leads to a decreased signal‐to‐noise ratio (lower panel) and impaired night vision in affected patients.