With the continual evolution of new strains severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The SARS-CoV-2 main protease (M

Abstract Coronaviruses can evolve and spread rapidly to cause severe disease morbidity mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such proteases polymerases, represent key classes antivirals. However, clinical use antiviral therapies inevitably leads emergence drug resistance. In this study...

The ssrA tag, an 11-aa peptide added to the C terminus of proteins stalled during translation, targets for degradation by ClpXP and ClpAP. Mutational analysis tag reveals independent, but overlapping determinants its interactions with ClpX, ClpA, SspB, a specificity-enhancing factor ClpX. ClpX interacts residues 9–11 at whereas ClpA recognizes positions 8–10 in addition 1–2 N terminus. SspB 1–4 7, N-terminal ClpX-binding determinants, determinants. As result, work together recognize...

Adaptor proteins help proteases modulate substrate choice, ensuring that appropriate are degraded at the proper time and place. SspB is an adaptor delivers ssrA-tagged to AAA+ protease ClpXP for degradation. To identify new SspB-regulated substrates, we examined captured by trap in sspB + but not - strains. RseA 1-108 , a fragment of transmembrane protein regulates extracytoplasmic-stress response, fits this criterion. In response stress, cleaved on each side membrane released as cytoplasmic...

Significance When mutations within a protein change each other’s functional effects—a phenomenon called epistasis—the paths available to evolution at any moment in time depend on the specific set of changes that previously occurred protein. The extent which epistasis has shaped historical evolutionary trajectories is unknown. Using high-precision bulk fitness assay and ancestral reconstruction, we measured effects extant sequences all substitutions during billion-year trajectory an essential...

To probe the mechanism of Hsp90 chaperone that is required for maturation many signaling proteins in eukaryotes, we analyzed effects all individual amino acid changes ATPase domain on yeast growth rate. The sensitivity a position to mutation was strongly influenced by proximity phosphates ATP, indicating ATPase-driven conformational impose stringent physical constraints Hsp90. investigate how these may vary different clients, performed biochemical analyses panel mutants spanning full range...

Gene-environment interactions have long been theorized to influence molecular evolution. However, the environmental dependence of most mutations remains unknown. Using deep mutational scanning, we engineered yeast with all 44,604 single codon changes encoding 14,160 amino acid variants in Hsp90 and quantified growth effects under standard conditions five stress conditions. To our knowledge, these are largest determined comprehensive fitness maps point mutants. The many differed between...

Drugs that target the main protease (Mpro) of SARS-CoV-2 are effective therapeutics have entered clinical use. Wide-scale use these drugs will apply selection pressure for evolution resistance mutations. To understand potential in Mpro, we performed comprehensive surveys amino acid changes can cause to nirmatrelvir (Pfizer), and ensitrelvir (Xocova) a yeast screen. We identified 142 mutations 177 ensitrelvir, many which not been previously reported. Ninety-nine caused apparent both...

Proteolytic cascades often transduce signals between cellular compartments, but the features of these that permit efficient conversion a biological signal into transcriptional output are not well elucidated. σ E mediates an envelope stress response in Escherichia coli , and its activity is controlled by regulated degradation RseA, membrane-spanning anti-σ factor. Examination individual steps this protease cascade reveals initial, signal-sensing cleavage step rate-limiting; multiple...

Most of the fundamental processes cells are mediated by proteins. However, biologically-relevant mechanism most proteins poorly understood. Dominant negative mutations have provided a valuable tool for investigating protein mechanisms but can be difficult to isolate because their toxic effects. We used mutational scanning approach identify dominant in yeast Hsp90. Hsp90 is chaperone that forms dynamic complexes with many co-chaperones and client In vitro analyses elucidated some key...

The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by the SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, resistance-conferring impact inhibitor binding or enzyme activity. Nirmatrelvir, most widely used currently to treat infections, targets main protease (Mpro) preventing it from processing viral polyprotein into active subunits. Our previous work systematically analyzed Mpro reduce...

The DNA-damage response genes in bacteria are up-regulated when LexA repressor undergoes autocatalytic cleavage stimulated by activated RecA protein. Intact is stable to intracellular degradation but its auto-cleavage fragments degraded rapidly. Here, both of shown be substrates for the ClpXP protease. recognizes these using sequence motifs that flank site dormant intact LexA. Furthermore, LexA-DNA-binding fragment important cell survival after DNA damage. These results demonstrate how one...

The SARS-CoV-2 main protease (M pro ) is essential for viral replication, and a primary target COVID-19 antivirals. Direct-acting antivirals such as nirmatrelvir, the active component of Paxlovid, M site to block polyprotein cleavage thus replication. However, drug resistance mutations at residue Glu166 (E166) have emerged in vitro selection studies, raising concerns about durability current antiviral strategies. Here, we investigate molecular basis conferred by E166A E166V against...

Allosteric regulation is central to protein function in cellular networks. A fundamental open question whether of allosteric proteins occurs only at a few defined positions or many sites distributed throughout the structure. Here, we probe GTPases—protein switches that control signaling through regulated conformational cycling—at residue-level resolution by deep mutagenesis native biological network. For GTPase Gsp1/Ran, find 28% 4,315 assayed mutations show pronounced gain-of-function...

With the spread of artemisinin resistance throughout South East Asia and now in Africa, antimalarial drug pyronaridine is likely to become an increasingly important component new regimens. However, activity humans has not been completely characterized. This volunteer infection study aimed determine pharmacokinetic/pharmacodynamic (PK/PD) relationship malaria naïve adults. Volunteers were inoculated with Plasmodium falciparum-infected erythrocytes on day 0 administered different single oral...

Phosducin (Pd), a small protein found abundantly in photoreceptors, is widely assumed to regulate light sensitivity the rod outer segment through interaction with heterotrimeric G transducin. But, based on histochemistry and Western blot analysis, Pd almost entirely inner both dark, most near synapse. We report second protein, 14-3-3, similar distribution. By immunoprecipitation, phospho-Pd interact 14-3-3 material from dark-adapted retina, this markedly diminished by light, which...

Abstract Drugs that target the main protease (M pro ) of SARS-CoV-2 are effective therapeutics have entered clinical use. Wide-scale use these drugs will apply selection pressure for evolution resistance mutations. To understand potential in M , we performed comprehensive surveys amino acid changes can cause a yeast screen to nirmatrelvir (contained drug Paxlovid), and ensitrelvir (Xocova) is currently phase III trials. The most impactful mutation (E166V) recently reported multiple viral...

Dominant negative mutations provide valuable tools for investigating protein mechanisms but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach identify dominant (DN) in yeast Hsp90. In previous scan the ATPase domain Hsp90, we noticed that many were at very low frequency after outgrowth cells co-expressing WT Most these depleted variants located hinge lid closes over ATP. To quantify effects regions, performed using an inducible promoter and...

The distribution of fitness effects (DFEs) new mutations across different environments quantifies the potential for adaptation in a given environment and its cost others. So far, results regarding have been mixed, most studies sampled random genes. Here, we quantify systematically how costs vary along large stretch protein sequence by studying same ≈2,300 amino-acid changing obtained from deep mutational scanning 119 amino acids middle domain heat shock Hsp90 five environments. This region...