Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Mutational biases favor complexity increases in protein interaction networks after gene duplication.

Biological systems can gain complexity over time. While some of these transitions are likely driven by natural selection, the extent to which they occur without providing an adaptive benefit is unknown. At the molecular level, one example is heteromeric complexes replacing homomeric ones following gene duplication. Here, we build a biophysical model and simulate the evolution of homodimers and heterodimers following gene duplication using distributions of mutational effects inferred from available protein structures. We keep the specific activity of each dimer identical, so their concentrations drift neutrally without new functions. We show that for more than 60% of tested dimer structures, the relative concentration of the heteromer increases over time due to mutational biases that favor the heterodimer. However, allowing mutational effects on synthesis rates and differences in the specific activity of homo- and heterodimers can limit or reverse the observed bias toward heterodimers. Our results show that the accumulation of more complex protein quaternary structures is likely under neutral evolution, and that natural selection would be needed to reverse this tendency.

Cryptic genetic variation shapes the fate of gene duplicates in a protein interaction network.

Paralogous genes are often redundant for long periods of time before they diverge in function. While their functions are preserved, paralogous proteins can accumulate mutations that, through epistasis, could impact their fate in the future. By quantifying the impact of all single-amino acid substitutions on the binding of two myosin proteins to their interaction partners, we find that the future evolution of these proteins is highly contingent on their regulatory divergence and the mutations that have silently accumulated in their protein binding domains. Differences in the promoter strength of the two paralogs amplify the impact of mutations on binding in the lowly expressed one. While some mutations would be sufficient to non-functionalize one paralog, they would have minimal impact on the other. Our results reveal how functionally equivalent protein domains could be destined to specific fates by regulatory and cryptic coding sequence changes that currently have little to no functional impact.

Compensatory mutations potentiate constructive neutral evolution by gene duplication.

Protein functions generally depend on their assembly into complexes. During evolution, some complexes have transitioned from homomers encoded by a single gene to heteromers encoded by duplicate genes. This transition could occur without adaptive evolution through intermolecular compensatory mutations. Here, we experimentally duplicate and evolve an homodimeric enzyme to examine if and how this could happen. We identify hundreds of deleterious mutations that inactivate individual homodimers but produce functional enzymes when co-expressed as duplicated proteins that heterodimerize. The structure of one such heteromer reveals how both losses of function are buffered through the introduction of asymmetry in the complex that allows them to subfunctionalize. Constructive neutral evolution can thus occur by gene duplication followed by only one deleterious mutation per duplicate.

The genomic landscape of transposable elements in yeast hybrids is shaped by structural variation and genotype-specific modulation of transposition rate.

Transposable elements (TEs) are major contributors to structural genomic variation by creating interspersed duplications of themselves. In return, structural variants (SVs) can affect the genomic distribution of TE copies and shape their load. One long-standing hypothesis states that hybridization could trigger TE mobilization and thus increase TE load in hybrids. We previously tested this hypothesis (Hénault et al., 2020) by performing a large-scale evolution experiment by mutation accumulation (MA) on multiple hybrid genotypes within and between wild populations of the yeasts Saccharomyces paradoxus and Saccharomyces cerevisiae. Using aggregate measures of TE load with short-read sequencing, we found no evidence for TE load increase in hybrid MA lines. Here, we resolve the genomes of the hybrid MA lines with long-read phasing and assembly to precisely characterize the role of SVs in shaping the TE landscape. Highly contiguous phased assemblies of 127 MA lines revealed that SV types like polyploidy, aneuploidy, and loss of heterozygosity have large impacts on the TE load. We characterized 18 de novo TE insertions, indicating that transposition only has a minor role in shaping the TE landscape in MA lines. Because the scarcity of TE mobilization in MA lines provided insufficient resolution to confidently dissect transposition rate variation in hybrids, we adapted an in vivo assay to measure transposition rates in various S. paradoxus hybrid backgrounds. We found that transposition rates are not increased by hybridization, but are modulated by many genotype-specific factors including initial TE load, TE sequence variants, and mitochondrial DNA inheritance. Our results show the multiple scales at which TE load is shaped in hybrid genomes, being highly impacted by SV dynamics and finely modulated by genotype-specific variation in transposition rates.

Dissection of the role of a Src homology 3 domain in the evolution of binding preference of paralogous proteins.

Protein-protein interactions (PPIs) drive many cellular processes. Some interactions are directed by Src homology 3 (SH3) domains that bind proline-rich motifs on other proteins. The evolution of the binding specificity of SH3 domains is not completely understood, particularly following gene duplication. Paralogous genes accumulate mutations that can modify protein functions and, for SH3 domains, their binding preferences. Here, we examined how the binding of the SH3 domains of 2 paralogous yeast type I myosins, Myo3 and Myo5, evolved following duplication. We found that the paralogs have subtly different SH3-dependent interaction profiles. However, by swapping SH3 domains between the paralogs and characterizing the SH3 domains freed from their protein context, we find that very few of the differences in interactions, if any, depend on the SH3 domains themselves. We used ancestral sequence reconstruction to resurrect the preduplication SH3 domains and examined, moving back in time, how the binding preference changed. Although the most recent ancestor of the 2 domains had a very similar binding preference as the extant ones, older ancestral domains displayed a gradual loss of interaction with the modern interaction partners when inserted in the extant paralogs. Molecular docking and experimental characterization of the free ancestral domains showed that their affinity with the proline motifs is likely not the cause for this loss of binding. Taken together, our results suggest that a SH3 and its host protein could create intramolecular or allosteric interactions essential for the SH3-dependent PPIs, making domains not functionally equivalent even when they have the same binding specificity.

Parallel Nonfunctionalization of CK1δ/ε Kinase Ohnologs Following a Whole-Genome Duplication Event.

Whole-genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in Saccharomyces cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post-WGD. In S. cerevisiae, HRR25 encodes the casein kinase 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post-WGD and non-WGD species, and have nonconserved cellular localization, consistent with their ongoing loss of function. The analysis in Naumovozyma castellii shows that the noncomplementing ohnolog is expressed at a lower level and has become nonessential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

Cross-feeding affects the target of resistance evolution to an antifungal drug.

Pathogenic fungi are a cause of growing concern. Developing an efficient and safe antifungal is challenging because of the similar biological properties of fungal and host cells. Consequently, there is an urgent need to better understand the mechanisms underlying antifungal resistance to prolong the efficacy of current molecules. A major step in this direction would be to be able to predict or even prevent the acquisition of resistance. We leverage the power of experimental evolution to quantify the diversity of paths to resistance to the antifungal 5-fluorocytosine (5-FC), commercially known as flucytosine. We generated hundreds of independent 5-FC resistant mutants derived from two genetic backgrounds from wild isolates of Saccharomyces cerevisiae. Through automated pin-spotting, whole-genome and amplicon sequencing, we identified the most likely causes of resistance for most strains. Approximately a third of all resistant mutants evolved resistance through a pleiotropic drug response, a potentially novel mechanism in response to 5-FC, marked by cross-resistance to fluconazole. These cross-resistant mutants are characterized by a loss of respiration and a strong tradeoff in drug-free media. For the majority of the remaining two thirds, resistance was acquired through loss-of-function mutations in FUR1, which encodes an important enzyme in the metabolism of 5-FC. We describe conditions in which mutations affecting this particular step of the metabolic pathway are favored over known resistance mutations affecting a step upstream, such as the well-known target cytosine deaminase encoded by FCY1. This observation suggests that ecological interactions may dictate the identity of resistance hotspots.

Parallel nonfunctionalization of CK1δ/ε kinase ohnologs following a whole-genome duplication event.

Whole genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in S. cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post WGD. In S. cerevisiae, HRR25 encodes the casein kinase (CK) 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post and non-WGD species, and have non-conserved cellular localization, consistent with their ongoing loss of function. The analysis in N. castelli shows that the non-complementing ohnolog is expressed at a lower level and has become non-essential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

The fitness cost of spurious phosphorylation.

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known, but quantifying this is required to understand the constraints faced by cell systems as they evolve. Here, we use the model organism S. cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, most of the resulting tyrosine phosphorylation is spurious. This provides a suitable system to measure the impact of artificial protein interactions on fitness. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3,500 proteins. Examination of the fitness costs in each strain revealed a strong correlation between the number of spurious pY sites and decreased growth. Moreover, the analysis of pY effects on protein structure and on protein function revealed over 1000 pY events that we predict to be deleterious. However, we also find that a large number of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with bona fide tyrosine kinases. Taken together, our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve.

A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics.

BACKGROUND: Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. RESULTS: We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. CONCLUSIONS: Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

Evolutionary trade-off and mutational bias could favor transcriptional over translational divergence within paralog pairs.

How changes in the different steps of protein synthesis-transcription, translation and degradation-contribute to differences of protein abundance among genes is not fully understood. There is however accumulating evidence that transcriptional divergence might have a prominent role. Here, we show that yeast paralogous genes are more divergent in transcription than in translation. We explore two causal mechanisms for this predominance of transcriptional divergence: an evolutionary trade-off between the precision and economy of gene expression and a larger mutational target size for transcription. Performing simulations within a minimal model of post-duplication evolution, we find that both mechanisms are consistent with the observed divergence patterns. We also investigate how additional properties of the effects of mutations on gene expression, such as their asymmetry and correlation across levels of regulation, can shape the evolution of paralogs. Our results highlight the importance of fully characterizing the distributions of mutational effects on transcription and translation. They also show how general trade-offs in cellular processes and mutation bias can have far-reaching evolutionary impacts.

Breaking spore dormancy in budding yeast transforms the cytoplasm and the solubility of the proteome.

The biophysical properties of the cytoplasm are major determinants of key cellular processes and adaptation. Many yeasts produce dormant spores that can withstand extreme conditions. We show that spores of Saccharomyces cerevisiae exhibit extraordinary biophysical properties, including a highly viscous and acidic cytosol. These conditions alter the solubility of more than 100 proteins such as metabolic enzymes that become more soluble as spores transit to active cell proliferation upon nutrient repletion. A key regulator of this transition is the heat shock protein, Hsp42, which shows transient solubilization and phosphorylation, and is essential for the transformation of the cytoplasm during germination. Germinating spores therefore return to growth through the dissolution of protein assemblies, orchestrated in part by Hsp42 activity. The modulation of spores' molecular properties are likely key adaptive features of their exceptional survival capacities.

Evolutionary Trajectories are Contingent on Mitonuclear Interactions.

Critical mitochondrial functions, including cellular respiration, rely on frequently interacting components expressed from both the mitochondrial and nuclear genomes. The fitness of eukaryotic organisms depends on a tight collaboration between both genomes. In the face of an elevated rate of evolution in mtDNA, current models predict that the maintenance of mitonuclear compatibility relies on compensatory evolution of the nuclear genome. Mitonuclear interactions would therefore exert an influence on evolutionary trajectories. One prediction from this model is that the same nuclear genome evolving with different mitochondrial haplotypes would follow distinct molecular paths toward higher fitness. To test this prediction, we submitted 1,344 populations derived from 7 mitonuclear genotypes of Saccharomyces cerevisiae to >300 generations of experimental evolution in conditions that either select for a mitochondrial function or do not strictly require respiration for survival. Performing high-throughput phenotyping and whole-genome sequencing on independently evolved individuals, we identified numerous examples of gene-level evolutionary convergence among populations with the same mitonuclear background. Phenotypic and genotypic data on strains derived from this evolution experiment identify the nuclear genome and the environment as the main determinants of evolutionary divergence, but also show a modulating role for the mitochondrial genome exerted both directly and via interactions with the two other components. We finally recapitulated a subset of prominent loss-of-function alleles in the ancestral backgrounds and confirmed a generalized pattern of mitonuclear-specific and highly epistatic fitness effects. Together, these results demonstrate how mitonuclear interactions can dictate evolutionary divergence of populations with identical starting nuclear genotypes.

Macroevolutionary diversity of traits and genomes in the model yeast genus Saccharomyces.

Species is the fundamental unit to quantify biodiversity. In recent years, the model yeast Saccharomyces cerevisiae has seen an increased number of studies related to its geographical distribution, population structure, and phenotypic diversity. However, seven additional species from the same genus have been less thoroughly studied, which has limited our understanding of the macroevolutionary events leading to the diversification of this genus over the last 20 million years. Here, we show the geographies, hosts, substrates, and phylogenetic relationships for approximately 1,800 Saccharomyces strains, covering the complete genus with unprecedented breadth and depth. We generated and analyzed complete genome sequences of 163 strains and phenotyped 128 phylogenetically diverse strains. This dataset provides insights about genetic and phenotypic diversity within and between species and populations, quantifies reticulation and incomplete lineage sorting, and demonstrates how gene flow and selection have affected traits, such as galactose metabolism. These findings elevate the genus Saccharomyces as a model to understand biodiversity and evolution in microbial eukaryotes.

Epistasis between promoter activity and coding mutations shapes gene evolvability.

The evolution of protein-coding genes proceeds as mutations act on two main dimensions: regulation of transcription level and the coding sequence. The extent and impact of the connection between these two dimensions are largely unknown because they have generally been studied independently. By measuring the fitness effects of all possible mutations on a protein complex at various levels of promoter activity, we show that promoter activity at the optimal level for the wild-type protein masks the effects of both deleterious and beneficial coding mutations. Mutations that are deleterious at low activity but masked at optimal activity are slightly destabilizing for individual subunits and binding interfaces. Coding mutations that increase protein abundance are beneficial at low expression but could potentially incur a cost at high promoter activity. We thereby demonstrate that promoter activity in interaction with protein properties can dictate which coding mutations are beneficial, neutral, or deleterious.

Hybridization drives mitochondrial DNA degeneration and metabolic shift in a species with biparental mitochondrial inheritance.

Mitochondrial DNA (mtDNA) is a cytoplasmic genome that is essential for respiratory metabolism. Although uniparental mtDNA inheritance is most common in animals and plants, distinct mtDNA haplotypes can coexist in a state of heteroplasmy, either because of paternal leakage or de novo mutations. mtDNA integrity and the resolution of heteroplasmy have important implications, notably for mitochondrial genetic disorders, speciation, and genome evolution in hybrids. However, the impact of genetic variation on the transition to homoplasmy from initially heteroplasmic backgrounds remains largely unknown. Here, we use Saccharomyces yeasts, fungi with constitutive biparental mtDNA inheritance, to investigate the resolution of mtDNA heteroplasmy in a variety of hybrid genotypes. We previously designed 11 crosses along a gradient of parental evolutionary divergence using undomesticated isolates of Saccharomyces paradoxus and Saccharomyces cerevisiae Each cross was independently replicated 48 to 96 times, and the resulting 864 hybrids were evolved under relaxed selection for mitochondrial function. Genome sequencing of 446 MA lines revealed extensive mtDNA recombination, but the recombination rate was not predicted by parental divergence level. We found a strong positive relationship between parental divergence and the rate of large-scale mtDNA deletions, which led to the loss of respiratory metabolism. We also uncovered associations between mtDNA recombination, mtDNA deletion, and genome instability that were genotype specific. Our results show that hybridization in yeast induces mtDNA degeneration through large-scale deletion and loss of function, with deep consequences for mtDNA evolution, metabolism, and the emergence of reproductive isolation.

Asymmetrical dose responses shape the evolutionary trade-off between antifungal resistance and nutrient use.

Antimicrobial resistance is an emerging threat for public health. The success of resistance mutations depends on the trade-off between the benefits and costs they incur. This trade-off is largely unknown and uncharacterized for antifungals. Here, we systematically measure the effect of all amino acid substitutions in the yeast cytosine deaminase Fcy1, the target of the antifungal 5-fluorocytosine (5-FC, flucytosine). We identify over 900 missense mutations granting resistance to 5-FC, a large fraction of which appear to act through destabilization of the protein. The relationship between 5-FC resistance and growth sustained by cytosine deamination is characterized by a sharp trade-off, such that small gains in resistance universally lead to large losses in canonical enzyme function. We show that this steep relationship can be explained by differences in the dose-response functions of 5-FC and cytosine. Finally, we observe the same trade-off shape for the orthologue of FCY1 in Cryptoccocus neoformans, a human pathogen. Our results provide a powerful resource and platform for interpreting drug target variants in fungal pathogens as well as unprecedented insights into resistance-function trade-offs.

Correlation between protein abundance and sequence conservation: what do recent experiments say?

Cells evolve in a space of parameter values set by physical and chemical forces. These constraints create associations among cellular properties. A particularly strong association is the negative correlation between the rate of evolution of proteins and their abundance in the cell. Highly expressed proteins evolve slower than lowly expressed ones. Multiple hypotheses have been put forward to explain this relationship, including, for instance, the requirement for higher mRNA stability, misfolding avoidance, and misinteraction avoidance for highly expressed proteins. Here, we review some of these hypotheses, their predictions, and how they are supported to finally discuss recent experiments that have been performed to test these predictions.