Analyses of the effects of all ubiquitin point mutants on yeast growth rate

Abstract

The amino acid sequence of a protein governs its function. We used bulk competition and focused deep sequencing to investigate the effects of all ubiquitin point mutants on yeast growth rate. Many aspects of ubiquitin function have been carefully studied, which enabled interpretation of our growth analyses in light of a rich structural, biophysical and biochemical knowledge base. In one highly sensitive cluster on the surface of ubiquitin, almost every amino acid substitution caused growth defects. In contrast, the opposite face tolerated virtually all possible substitutions. Surface locations between these two faces exhibited intermediate mutational tolerance. The sensitive face corresponds to the known interface for many binding partners. Across all surface positions, we observe a strong correlation between burial at structurally characterized interfaces and the number of amino acid substitutions compatible with robust growth. This result indicates that binding is a dominant determinant of ubiquitin function. In the solvent-inaccessible core of ubiquitin, all positions tolerated a limited number of substitutions, with hydrophobic amino acids especially interchangeable. Some mutations null for yeast growth were previously shown to populate folded conformations indicating that, for these mutants, subtle changes to conformation caused functional defects. The most sensitive region to mutation within the core was located near the C-terminus that is a focal binding site for many critical binding partners. These results indicate that core mutations may frequently cause functional defects through subtle disturbances to structure or dynamics.

Figure 1

Bulk competition analyses of the effect of ubiquitin mutants on yeast growth. (a) Experimental setup: systematic libraries of ubiquitin point mutants generated using saturation mutagenesis at sequential positions within a 9–10 amino acid window were generated on a plasmid with a constitutive promoter. These libraries were introduced into a ubiquitin strain whose only other source of ubiquitin was regulated by a galactose dependent promoter. Yeast libraries were amplified in galactose, and then competed in dextrose where growth relied on the mutant ubiquitin library. (b) Growth of the ubiquitin shutoff strain is rescued by constitutive expression of WT ubiquitin. (c) Positions 40–48 of ubiquitin were selected for initial method development. (d–e) Sequence based analyses of bulk competition of libraries of ubiquitin point mutants at positions 40–48. (d) Stop codons were rapidly depleted indicating that they were unable to support growth, while silent substitutions that change the nucleotide sequence without altering the protein sequence persisted in shutoff conditions. (e) Correlation between measured growth effects of mutants (selection coefficient) from full experimental repeats.

Figure 2

Analyses of the growth effects of mutants across the ubiquitin gene. (a) The gene was subdivided into eight regions of 9–10 amino acids and each region was subject to saturation mutagenesis, bulk competition in yeast, and deep sequencing analyses. (b) Heat map representation of the effects of ubiquitin mutants on yeast growth. Mutants that were below a conservative detection limit at the beginning of the competition were omitted from fitness analyses. (c) Bi-modal distribution of observed mutant effects on yeast growth indicates that most mutants supported either WT-like or null growth in yeast (d) Distribution of growth effects for mutations that depleted by more than 2-fold during outgrowth in galactose media, but remained sufficiently abundant to quantify fitness. Most depleted mutants had null-like fitness and none were WT-like (s>−0.1). (e) Correlation between the growth rate of a panel of individually analyzed mutants relative to fitness measures from bulk competitions.

Figure 3

Effects of mutants on the solvent-accessible surface of ubiquitin on yeast growth. (a) Distribution of the number of amino acids observed to support growth within 90% or greater of wild type ubiquitin. Many positions in ubiquitin are either highly sensitive to mutation (4 or less amino acids support robust growth), or highly tolerant (17 or more amino acids support robust growth). (b) Space filling representations of ubiquitin structure (based on 1UBQ.PDB) with sensitive positions colored blue, tolerant yellow, and intermediate green. (c) Heat map representations of sensitive, intermediate and tolerant positions on the ubiquitin surface. One-dimensional maps on the bottom compare our analyses with a previous alanine scan. At positions where the WT amino acid is alanine, glycine substitutions are shown for both the EMPIRIC and previous alanine scan.

Figure 4

Relating fitness sensitivity on the surface of ubiquitin to binding interfaces. Structural representations of ubiquitin bound to common binding domains: (a) UBA domain (2OOB.PDB), and (b) UIM domain (1QOW.PDB). Top images show binding domains in magenta and ubiquitin as space-filled spheres with fitness sensitive positions in blue, fitness tolerant positions in yellow, and intermediate positions in green. Bottom images illustrate the underlying ubiquitin secondary structure. (c) Fraction of surface area buried per sensitive or tolerant residue on the surface of ubiquitin in 44 high-resolution co-crystal structures. Error bars represent standard deviations (N=18 and 19 respectively) (d) Correlation between fitness tolerance to amino acid substitution and burial at structurally characterized interfaces.

Figure 5

Relating fitness sensitivity to structure of tetra-ubiquitin (a) Structural image of K48-linked tetra-ubiquitin (2O6V.PDB). Top images show space-filling representation with fitness sensitive positions colored blue, tolerant positions yellow, and intermediate positions grey. Different color shades were used to distinguish subunits. Bottom image illustrates the underlying secondary structure. (b) Fractoin surface area buried per sensitive or tolerant residue in tetra-ubiquitin. Error bars represent standard deviations (N=18 and 19 respectively)

Figure 6

Mutant effects in the solvent-inaccessible core of ubiquitin. (a) Heat map indicating the fitness of mutations at core positions indicates that substitutions among aliphatic amino acids are generally well-tolerated. (b) Positions in the core exhibit an intermediate tolerance to mutation with most positions having 3–6 different amino acids that support growth rates similar to the wild type sequence (s>−0.1). (c) Structural representation of ubiquitin showing the wild type side chains of core positions. Positions that tolerate more than eight amino acids (s>−0.1) are colored in yellow. (d) Relationship between core mutant impacts on folding stability^; and yeast growth. Previously measured effects on ΔΔG of folding are plotted such that negative numbers represent destabilization. The amount of destabilization estimated to abolish folding is indicated as a dashed grey line on the left. Mutations to Q41, the only WT core polar amino acid, are shown in grey. All mutations in this panel are estimated to populate the unfolded state less than 1% in the absence of elevated temperature or denaturant based on the stability of wild type ubiquitin. Mutants that are destabilized by more than 2 kcal/mol are shown in orange if they are highly fit (s>−0.12), blue if they are strongly deleterious (s<−0.49), or purple for those with intermediate fitness. (e) Structure of ubiquitin indicating the location of destabilized and highly fit (yellow) as well as destabilized and deleterious (cyan) mutations.