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Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.
- Source :
-
PLoS genetics [PLoS Genet] 2024 Apr 29; Vol. 20 (4), pp. e1011252. Date of Electronic Publication: 2024 Apr 29 (Print Publication: 2024). - Publication Year :
- 2024
-
Abstract
- 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.<br />Competing Interests: The authors have declared that no competing interests exist.<br /> (Copyright: © 2024 Rouleau et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Subjects :
- Allosteric Regulation
Saccharomyces cerevisiae genetics
Saccharomyces cerevisiae drug effects
Humans
Fungal Proteins genetics
Fungal Proteins metabolism
Fungal Proteins chemistry
Catalytic Domain genetics
Tetrahydrofolate Dehydrogenase genetics
Tetrahydrofolate Dehydrogenase metabolism
Tetrahydrofolate Dehydrogenase chemistry
Pneumocystis carinii genetics
Pneumocystis carinii enzymology
Pneumocystis carinii drug effects
Folic Acid Antagonists pharmacology
Drug Resistance, Fungal genetics
Mutation
Methotrexate pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 1553-7404
- Volume :
- 20
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- PLoS genetics
- Publication Type :
- Academic Journal
- Accession number :
- 38683847
- Full Text :
- https://doi.org/10.1371/journal.pgen.1011252