Identification of three distinct classes of XLRS mutations
The identification of complex interfaces using PDBePISA analysis (Supplementary Material, Fig. S8) allowed for interpretation of the effect of mutations upon the assembled retinoschisin molecule. Non-cysteine XLRS-associated mutations which caused conservative changes or were previously predicted to have negligible effects on monomer folding (29,30) were mapped onto the structure (Supplementary Material, Fig. S9). One identified class of mutations clustered at the intra-octamer interface (Supplementary Material, Fig. S9A). Indeed, E72, N104 and T185 were implicated in direct contact between the domains (Supplementary Material, Fig. S9), with both E72K and T185K mutations retained intracellularly (21,22). Another class of conservative mutations was located at the inter-octamer interface. Two contact sites were previously suggested (27) and here an additional contact site is identified. This third site between the octamer rings is formed by the loop between strands β4 and β5 (residues 178–182). All three contact sites have conservative mutations which lead to XLRS, these include residues in strand β4 (D145 and E146), the proximal loop region (G178, and N179) as well as strand β7 (H207 and R209) (Supplementary Material, Fig. S9B).
Mapping of the R141H mutation indicated that it was located in a spike region distinct from intra- and inter-octamer interfaces (Fig. 6A) with an overall positive charge (Fig. 6B). Comparison of the intrinsic fluorescence of wild-type and R141H mutant retinoschisin showed increased fluorescence in the mutant despite the lack of gross conformational change (Fig. 6C), suggesting a small change in the spike regions. Indeed, analysis of the homology model revealed two buried tryptophan residues in the model (W112 and W147) found in spikes 2 and 3 respectively, within 1 nm of the mutation site (Fig. 6D). This suggests the R141H mutation leads to a subtle conformational change within this region, however lowered local resolution in the electron density maps precluded observation of this alteration. Additionally, R141H has discrete bands visualized by native-PAGE (Fig. 5A) whereas the wild-type and H207Q mutant run as diffuse broad bands (Figs. 2A and 3B) suggesting that the R141H mutant changes the charge or conformation of the complex leading to altered mobility in native-PAGE. Together these subtle alterations may disrupt a binding interface within the propeller tips (Fig. 6E).
Figure 6. Mapping XLRS-causative conservative mutations onto the quasi-atomic model. (A) The position of residue R141 within the octamer is highlighted in red. (B) Electrostatic surface potential of the face of the discoidin domain containing residue R141 (circled) with overall positive charge. Scale bar for (A) and (B) = 25 nm. (C) Intrinsic fluorescence comparison of wild-type and R141H retinoschisin monomer. The R141H mutant has reduced fluorescence quenching suggesting increased solvent exposure of tryptophans. Shown is the emission spectrum after excitation at 295nm (n = 3). (D) Solvent accessible surface area (SASA) analysis of the tryptophans in the retinoschisin discoidin domain. W147 and W112 are buried and close to the R141H mutation site. (E) The paired octamer model for retinoschisin structural support between photoreceptor and bipolar cell synaptic membranes, with the sites affected by R141H and H207Q mutations labelled.