Saturation mutagenesis screen to identify compensatory mutants that rescue RNA-binding activity of ProQR80K. (A) Residues selected for saturation mutagenesis are highlighted in yellow, green, and cyan on the NMR structure of ProQNTD (Gonzalez et al. 2017). Arginine 80 is shown in orange sticks in the convex-face position found in the NMR structure and in pink sticks in a hypothetical concave-face position analogous to its position in other orthologs. (B) Compensatory mutations found in pPrey-ProQR80K plasmids that produced blue colonies on X-gal-indicator plates with pBait-malM-3′ are listed. Codons identified in multiple colonies are indicated by 2× and 3×. (C) Results confirming the effects of compensatory mutants in a plate-based B3H assay, detecting interactions between variants of ProQΔCTD with malM-3′ RNA. β-gal assays were performed with Δhfq reporter strain cells containing three compatible plasmids: one that encoded the CI-MS2CP fusion protein, another that encoded α or an α-ProQΔCTD fusion protein (wild-type, WT, or the indicated mutant), and a third that encoded a hybrid RNA (MS2hp-malM-3′) (see Materials and Methods). Quantification of color intensity shown in Supplemental Figure S4B. (D,E) The two strongest compensatory substitutions (V74K and L91V) are shown on (D) the NMR structure and (E) the AlphaFold Model (Varadi et al. 2022) of ProQNTD. Side chains were mutated in PyMol to the lowest-energy rotamer to show predicted structures of the compensatory substitutions. Distances between the terminal atom of each side chain (R80K, V74K, L71V) are shown in Angstroms and visualized with dashed lines.
