Abstract
Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available ...vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (M
pro
) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 M
pro
bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of M
pro
, map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed M
pro
inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.
Experimental illumination of a fitness landscape Hietpas, Ryan T; Jensen, Jeffrey D; Bolon, Daniel N.A
Proceedings of the National Academy of Sciences - PNAS,
05/2011, Letnik:
108, Številka:
19
Journal Article
Recenzirano
Odprti dostop
The genes of all organisms have been shaped by selective pressures. The relationship between gene sequence and fitness has tremendous implications for understanding both evolutionary processes and ...functional constraints on the encoded proteins. Here, we have exploited deep sequencing technology to experimentally determine the fitness of all possible individual point mutants under controlled conditions for a nine-amino acid region of Hsp90. Over the past five decades, limited glimpses into the relationship between gene sequence and function have sparked a long debate regarding the distribution, relative proportion, and evolutionary significance of deleterious, neutral, and advantageous mutations. Our systematic experimental measurement of fitness effects of Hsp90 mutants in yeast, evaluated in the light of existing population genetic theory, are remarkably consistent with a nearly neutral model of molecular evolution.
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.
Display omitted
► Mutations provide a powerful probe of protein mechanism. ► Bulk competition and deep sequencing was used to monitor ubiquitin mutants. ► Sensitivity to mutation correlated with binding interfaces at surface positions. ► In the core, positions near critical binding sites were the most sensitive to mutation. ► Binding interactions impose dominant binding constraints throughout ubiquitin.
Mutations are the source of evolutionary variation. The interactions of multiple mutations can have important effects on fitness and evolutionary trajectories. We have recently described the ...distribution of fitness effects of all single mutations for a nine-amino-acid region of yeast Hsp90 (Hsp82) implicated in substrate binding. Here, we report and discuss the distribution of intragenic epistatic effects within this region in seven Hsp90 point mutant backgrounds of neutral to slightly deleterious effect, resulting in an analysis of more than 1,000 double mutants. We find negative epistasis between substitutions to be common, and positive epistasis to be rare--resulting in a pattern that indicates a drastic change in the distribution of fitness effects one step away from the wild type. This can be well explained by a concave relationship between phenotype and genotype (i.e., a concave shape of the local fitness landscape), suggesting mutational robustness intrinsic to the local sequence space. Structural analyses indicate that, in this region, epistatic effects are most pronounced when a solvent-inaccessible position is involved in the interaction. In contrast, all 18 observations of positive epistasis involved at least one mutation at a solvent-exposed position. By combining the analysis of evolutionary and biophysical properties of an epistatic landscape, these results contribute to a more detailed understanding of the complexity of protein evolution.
The last decade has seen a growing number of experiments aimed at systematically mapping the effects of mutations in different proteins, and of attempting to correlate their biophysical and ...biochemical effects with organismal fitness. While insightful, systematic laboratory measurements of fitness effects present challenges and difficulties. Here, we discuss the limitations associated with such measurements, and in particular the challenge of correlating the effects of mutations at the single protein level (“protein fitness”) with their effects on organismal fitness. A variety of experimental setups are used, with some measuring the direct effects on protein function and others monitoring the growth rate of a model organism carrying the protein mutants. The manners by which fitness effects are calculated and presented also vary, and the conclusions, including the derived distributions of fitness effects of mutations, vary accordingly. The comparison of the effects of mutations in the laboratory to the natural protein diversity, namely to amino acid changes that have fixed in the course of millions of years of evolution, is also debatable. The results of laboratory experiments may, therefore, be less relevant to understanding long‐term inter‐species variations yet insightful with regard to short‐term polymorphism, for example, in the study of the effects of human SNPs.
The complexity of biological interaction networks poses a challenge to understanding the function of individual connections in the overall network. To address this challenge, we developed a ...high-throughput reverse engineering strategy to analyze how thousands of specific perturbations (encompassing all point mutations in a central gene) impact both a specific edge (interaction to a directly connected node) and an overall network function. We analyzed the effects of ubiquitin mutations on activation by the E1 enzyme and compared these to effects on yeast growth rate. Using this approach, we delineated ubiquitin mutations that selectively impacted the ubiquitin-E1 edge. We find that the elasticity function relating the efficiency of ubiquitin-E1 interaction to growth rate is non-linear and that a greater than 50-fold decrease in E1 activation efficiency is required to reduce growth rate by 2-fold. Despite the robustness of fitness to decreases in E1 activation efficiency, the effects of most ubiquitin mutations on E1 activation paralleled the effects on growth rate. Our observations indicate that most ubiquitin mutations that disrupt E1 activation also disrupt other functions. The structurally characterized ubiquitin-E1 interface encompasses the interfaces of ubiquitin with most other known binding partners, and we propose that this enables E1 in wild-type cells to selectively activate ubiquitin protein molecules capable of binding to other partners from the cytoplasmic pool of ubiquitin protein that will include molecules with chemical damage and/or errors from transcription and translation.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Deep sequencing can accurately measure the relative abundance of hundreds of mutations in a single bulk competition experiment, which can give a direct readout of the fitness of each mutant. Here we ...describe a protocol that we previously developed and optimized to measure the fitness effects of all possible individual codon substitutions for 10-aa regions of essential genes in yeast. Starting with a conditional strain (i.e., a temperature-sensitive strain), we describe how to efficiently generate plasmid libraries of point mutants that can then be transformed to generate libraries of yeast. The yeast libraries are competed under conditions that select for mutant function. Deep-sequencing analyses are used to determine the relative fitness of all mutants. This approach is faster and cheaper per mutant compared with analyzing individually isolated mutants. The protocol can be performed in ∼4 weeks and many 10-aa regions can be analyzed in parallel.
Interactions among mutations within a protein have the potential to make molecular evolution contingent and irreversible, but the extent to which epistasis actually shaped historical evolutionary ...trajectories is unclear. To address this question, we experimentally measured how the fitness effects of historical sequence substitutions changed during the billion-year evolutionary history of the heat shock protein 90 (Hsp90) ATPase domain beginning from a deep eukaryotic ancestor to modern Saccharomyces cerevisiae. We found a pervasive influence of epistasis. Of 98 derived amino acid states that evolved along this lineage, about half compromise fitness when introduced into the reconstructed ancestral Hsp90. And the vast majority of ancestral states reduce fitness when introduced into the extant S. cerevisiae Hsp90. Overall, more than 75% of historical substitutions were contingent on permissive substitutions that rendered the derived state nondeleterious, became entrenched by subsequent restrictive substitutions that made the ancestral state deleterious, or both. This epistasis was primarily caused by specific interactions among sites rather than a general effect on the protein’s tolerance to mutation. Our results show that epistasis continually opened and closed windows of mutational opportunity over evolutionary timescales, producing histories and biological states that reflect the transient internal constraints imposed by the protein’s fleeting sequence states.
Hsp90 is a homodimeric ATPase that is essential in eukaryotes for the maturation of client proteins frequently involved in signal transduction, including many kinases and nuclear steroid hormone ...receptors. Competitive inhibitors of ATP binding to Hsp90 prevent client maturation and show promise as anticancer agents in clinical trials. However, the role of ATP binding and hydrolysis in each subunit of the Hsp90 dimer has been difficult to investigate because of an inability to assemble and study dimers of defined composition. We used protein engineering to generate functional Hsp90 subunits that preferentially assemble as heterodimers. We analyzed dimers wherein one subunit harbors a disruptive mutation and observed that ATP binding by both subunits is essential for function in yeast, whereas ATP hydrolysis is only required in one subunit. These findings demonstrate important functional contributions from both symmetric and asymmetric Hsp90 dimers and provide valuable reagents for future investigations of Hsp90 mechanism.
Display omitted
•Protein engineering transforms Hsp90 from a homodimer to a functional heterodimer•ATP binding is essential in both Hsp90 subunits•ATP hydrolysis is required in only one Hsp90 subunit•Symmetric and asymmetric Hsp90 subunit conformations are important for function.
Hsp90 substrates are disproportionately linked to signaling. Inhibitors of ATP binding show therapeutic promise in cancer, yet the mechanism linking ATP hydrolysis to function is poorly understood. Mishra and Bolon find that the efficient function of Hsp90 homodimers requires ATP binding in both subunits but hydrolysis in only one subunit.
PDF
