Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance

Abstract

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl₂, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

Keywords: Deep mutational scanning; Evolution; Ubiquitin.

Fig. 1.

The difference in fitness between DMSO and a perturbation for each Ub allele. Chemical names are colored based on the hierarchical clustering presented in Fig. 1: (A) Cobalt, (B) p-FP, (C) Nickel, (D) 3-AT, (E) CaCl₂, (F) 5-FC, (G) Tunicamycin, (H) Cerulenin, (I) Menadione, (J) Rapamycin, (K) Tamoxifen, (L) AmpB. Difference in fitness is represented from 0.25 (Blue) to −0.25 (Red) with white representing no change from DMSO. Wild-type amino acids are shown in green and mutations without fitness values (due to lack of barcode or competition sequencing reads) are shown in grey.

Fig. 2.

Hierarchical clustering of the fitnesses reveals four distinct clusters. Treatment with Cobalt and p-FP (magenta) cluster together and close to the previously described ‘sensitizing treatments’ (Mavor et al., 2016), DTT, Caffeine and HU (red). Treatment with Menadione, Cerulenin, Tunicamycin and 5-FC cluster with DMSO and the previously described ‘alleviating treatment’ MG132 (blue).

Fig. 3.

New perturbations reveal constraints on all but two Ub positions. (A) The minimum average fitness of each position was calculated in: (top) DMSO, Caffeine, DTT, HU and MG132 and (bottom) in all conditions. Minimum average fitness was determined by calculating the average fitness of each position in each condition and taking the minimum value. Positions were binned into tolerant (≥−0.075 - Blue), intermediate (<−0.075 to >−0.35 - Pink) and sensitive (≤−0.35 - Red) and the distributions plotted. Calculating the minimum average fitness reveals how the new perturbations reveal additional constraints on the Ub fitness landscape. (B) Minimum average fitness score in: (left) DMSO, Caffeine, DTT, HU, and MG132 and (right) in all conditions mapped onto the Ub structure. C-alpha atoms are shown in spheres and the residues are colored according to average fitness. Met1 is colored grey. Treatment with Nickel, 3-AT and CaCl₂ cluster together (cyan) and close to the ‘alleviating treatment’ cluster. Treatment with AmpB, Rapamycin and Tamoxifen appear as outliers in this clustering (grey). The clustering was performed using euclidean distance between the vectors and used Ward's method to join the clusters. Clusters are colored based on the treatments being within 6 distance of each other.

Fig. 4.

Principal component analysis reveals specific signals related to K63 incorporation. (A) The first two principal components reveal a sensitizing cluster with negative values in each PC and an alleviating cluster in the center of the plot. Treatment with 3-AT, 5-FC or CaCl₂ appear between clusters with positive values in PC1 and negative values in PC2. The points are colored based on the hierarchical clustering shown in Fig. 1. (B) The contribution of each mutation to each principal component was visualized as a heat map. The percentage of the maximum contribution to that principal component is represented from 75% (Blue) to −75% (Red). PC1 is related to the general sensitivity of Ub mutants to perturbation. Regions with large positive contributions to PC1 correspond to the regions with increased mutational sensitivity in the sensitizing treatments. Strikingly this is coupled to negative contributions to PC1 for some mutations at the core residue Phe45 (top). PC2 differentiates ‘Sensitive Face’ residues (positive contributions) from ‘Tolerant Face’ residues (Negative Contributions) (middle). PC3 reveals mutations that are correlated with sensitization of Lys63 (bottom). (C) The average contribution of each mutation to a PC at each position was plotted from 75% (Blue) to −75% (Red) on the Ub monomer structure for PC1 (A), PC2 (B), and PC3 (C). (D) Rosetta ΔΔG calculations revealed that mutations that strongly destabilize the donor Ub pose on the MMS/Ubc13 (2GMI) heterodimer are localized to Lys11 and Pro37 (shown in sticks). In the case of Lys11, all mutations other than that to Arg destabilize the interface suggesting a salt bridge between Lys11 and Glu65 of Ubc13 (shown in green sticks) ubiquitin residues are colored by the contribution to PC3 as in panel C.