Latent effects of Hsp90 mutants revealed at reduced expression levels

Abstract

In natural systems, selection acts on both protein sequence and expression level, but it is unclear how selection integrates over these two dimensions. We recently developed the EMPIRIC approach to systematically determine the fitness effects of all possible point mutants for important regions of essential genes in yeast. Here, we systematically investigated the fitness effects of point mutations in a putative substrate binding loop of yeast Hsp90 (Hsp82) over a broad range of expression strengths. Negative epistasis between reduced expression strength and amino acid substitutions was common, and the endogenous expression strength frequently obscured mutant defects. By analyzing fitness effects at varied expression strengths, we were able to uncover all mutant effects on function. The majority of mutants caused partial functional defects, consistent with this region of Hsp90 contributing to a mutation sensitive and critical process. These results demonstrate that important functional regions of proteins can tolerate mutational defects without experimentally observable impacts on fitness.

Figure 1. Fitness effects of Hsp90 amino acid substitutions.

(A) The fitness effects of all possible amino acid substitutions were analyzed for the region highlighted in cyan. This representation is based on the crystal structure of yeast Hsp90 . (B) At endogenous expression strength, the distribution of fitness effects was bimodal with many mutations resulting in either WT-like (green) or null-like (red) growth rates.

Figure 2. Effect of reduced Hsp90 expression on yeast growth.

(A) Growth of Hsp90 shutoff yeast harboring rescue Hsp90 plasmids with varied promoters with or without a terminator in the 3′ UTR. Yeast were grown at 30°C and monitored by optical density at 600 nm. (B) Hsp90 expression in cells grown in dextrose for 19 hours was monitored by Western blotting. (C) Relationship between observed Hsp90 expression level and growth rate.

Figure 3. Mutant effects on protein function can be hidden to fitness analyses.

Fitness effects (top panel) are a property of the elasticity relationship between net protein function and growth (middle panel) and the impact of mutations on function per molecule (bottom panels). For Hsp90, the non-linear elasticity relationship could mask defects, making multiple distributions of functional effects (bottom panels) indistinguishable to fitness analyses. In the top and bottom panels, green represents mutants with WT-like, red null-like, and grey intermediate fitness effects.

Figure 4. Distribution of observed fitness effects.

EMPIRIC results for Hsp90 mutants with varied promoters with and without terminator sequences in the 3′ UTR: (A) TEF promoter, (B) TEF promoter without a terminator, (C) CYC promoter, (D) CYC promoter without a terminator, (E) ADH promoter, and (F) ADH promoter without a terminator.

Figure 5. Effects of mutations on Hsp90 function.

(A) Distribution of mutant effects on Hsp90 function calculated from fitness effects at varied expression levels. (B) Impact of mutations at two solvent exposed hydrophobic amino acids in Hsp90 on yeast growth at different expression strengths. (C) Mutant impacts on folding stability (−ΔΔG estimated from structural simulation) related to function. (D) Similarity of amino acid substitutions to wild type (based on Blosum62 matrix) relative to observed functional effects.

Figure 6. Expression level and stability of five non-conservative mutations.

(A) Expression level in shutoff yeast analyzed by Western blotting. The vertical line represents intervening lanes that were removed for clarity. (B) Secondary structure of purified C-domain constructs analyzed by circular dichroism. The spectrum of a denatured sample in 5M urea is shown for comparison (below 218 nm absorbance from urea interferes with signal). (C) Urea induced unfolding of purified C-domain constructs. The fraction unfolded was determined based on ellipticity at 222 nm.