Deep mutational scan of a drug efflux pump reveals its structure–function landscape

Meier, Gianmarco; Thavarasah, S.; Ehrenbolger, Kai; Hutter, Cedric A. J.; Hürlimann, Lea M.; Barandun, Jonas; Seeger, Markus A.

doi:10.1038/s41589-022-01205-1

Cited by 19 publications

(10 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The differential Cit uptake rates of the single and double mutants showcase how in vivo experimentally generated single mutations can alter the substrate spectrum of the yeast amino acid transporters without the loss of ancestral functions. Our observations are in line with studies of substrate promiscuity being the result of adaptive mutations in different membrane transporters [42–48]. The altered specificity for a substrate, which is mirrored in our case in the altered fitness, could be a result of a different affinity constant for the amino acid and/or maximal transport rate.…”

Section: Discussionsupporting

confidence: 89%

“…Our observations are in line with studies of substrate promiscuity being the result of adaptive mutations in different membrane transporters. [39]–[45] The altered specificity for a substrate, which is mirrored in our case in the altered fitness, could be a result of a different affinity constant for the amino acid and/or maximal transport rate.…”

Section: Discussionmentioning

confidence: 94%

See 1 more Smart Citation

Fitness landscape of substrate-adaptive mutations in evolved APC transporters

Karapanagioti,

Atlason,

Slotboom

et al. 2023

Preprint

View full text Add to dashboard Cite

The emergence of new protein functions is crucial for the evolution of organisms. This process has been extensively researched for soluble enzymes, but it is largely unexplored for membrane transporters, even though the ability to acquire new nutrients from a changing environment requires evolvability of transport functions. Here, we demonstrate the importance of environmental pressure in obtaining a new activity or altering a promiscuous activity in members of the Amino acid-Polyamine-organoCation (APC)-type yeast amino acid transporters family. We identify APC members that have broader substrate spectra than previously described. Usingin vivoexperimental evolution, we evolve two of these transporter genes,AGP1andPUT4, towards new substrate specificities. Single mutations on these transporters are found to be sufficient for expanding the substrate range of the proteins, while retaining the capacity to transport all original substrates. Nonetheless, each adaptive mutation comes with a distinct effect on the fitness for each of the original substrates, illustrating a trade-off between the ancestral and evolved functions. Collectively, our findings reveal how substrate-adaptive mutations in membrane transporters contribute to fitness and provide insights into how organisms can use transporter evolution to explore new ecological niches.

show abstract

Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 94%

Fitness landscape of substrate-adaptive mutations in evolved APC transporters

Karapanagioti,

Atlason,

Slotboom

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…CRISPR-based genome editing methods have become highly efficient and versatile in their application. They enabled the construction of thousands of targeted genomic edits in large mutant libraries, which were then screened for various phenotypes such as responses to antibiotics (Dewachter et al, 2023;Meier et al, 2022;Garst et al, 2017) or improved production of biochemicals (Liang et al, 2017). Here, we used CRISPR-based genome editing to screen for single amino acid changes that cause temperature-sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Mapping temperature-sensitive mutations at a genome-scale to engineer growth-switches inE. coli

Schramm

Pahl

Link

2023

Preprint

View full text Add to dashboard Cite

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of non-growing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate-metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D) produced homoserine for 24 hours, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with non-growing cells.

show abstract

“…a comprehensive and unbiased fashion. As demonstrated by recent applications (33)(34)(35)(36)(37)(38), DMS analysis readily reveals both the location and sequence variability of allostery hotspots, leading to new insights into the unique characteristics of allostery network in various systems. However, DMS studies are only able to identify phenotypes through the binary functional readout due to its high-throughput nature.…”

Section: R a F Tmentioning

confidence: 99%

Modulation of Allostery with Multiple Mechanisms by Hotspot Mutations in TetR

Deng,

Yuan,

Cui

2023

Preprint

View full text Add to dashboard Cite

For many biomedical and biotechnological applications, modulating allosteric coupling offers unique opportunities. Such efforts can largely benefit from efficient prediction and evaluation of allostery hotspot residues that dictate the degree of co-operativity between distant sites. In several hotspot mutants revealed by recent deep mutational scanning (DMS) experiments of a bacterial transcription factor, tetracycline repressor (TetR), we demonstrate that effects of allostery hotspot mutations can be evaluated by judiciously combining extensive unbiased and enhanced sampling molecular dynamics simulations. The results recapitulate the qualitative effects of these mutations on abolishing the induction function of TetR and provide a semi-quantitative rationale for the different degrees of rescuability to restore allosteric coupling of the hotspot mutations observed in the DMS analysis. Free energy landscapes and variations of structural and energetic properties of different functional states of the mutants show that the hotspot mutations perturb inter-domain coupling and/or intra-domain conformational properties relative to the wild type. Several mutations clearly perturb multiple properties, also highlighting the complexity of realistic situations. Thus, the results support our mechanistic model that hotspot residues contribute to allostery through distinct molecular mechanisms, a feature that we propose to be broadly applicable to many allosteric systems. Overall, our study provides general computational strategies for the identification and evaluation of hotspot mutations modulating allostery in proteins.

show abstract

Deep mutational scan of a drug efflux pump reveals its structure–function landscape

Cited by 19 publications

References 60 publications

Fitness landscape of substrate-adaptive mutations in evolved APC transporters

Fitness landscape of substrate-adaptive mutations in evolved APC transporters

Mapping temperature-sensitive mutations at a genome-scale to engineer growth-switches inE. coli

Modulation of Allostery with Multiple Mechanisms by Hotspot Mutations in TetR

Contact Info

Product

Resources

About