An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening
Enzyme I (EI), which is the key enzyme to activate the bacterial phosphotransferase system, plays an important role in the regulation of several metabolic pathways and controls the biology of bacterial cells at multiple levels. The conservation and ubiquity of EI among different types of bacteria ma...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-01-01
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| Series: | Journal of Structural Biology: X |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2590152420300167 |
| _version_ | 1818952741483970560 |
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| author | Trang T. Nguyen Vincenzo Venditti |
| author_facet | Trang T. Nguyen Vincenzo Venditti |
| author_sort | Trang T. Nguyen |
| collection | DOAJ |
| description | Enzyme I (EI), which is the key enzyme to activate the bacterial phosphotransferase system, plays an important role in the regulation of several metabolic pathways and controls the biology of bacterial cells at multiple levels. The conservation and ubiquity of EI among different types of bacteria makes the enzyme a potential target for antimicrobial research. Here, we use NMR-based fragment screening to identify novel inhibitors of EI. We identify three molecular fragments that allosterically inhibit the phosphoryl transfer reaction catalyzed by EI by interacting with the enzyme at a surface pocket located more than 10 Å away from the substrate binding site. Interestingly, although the three molecules share the same binding pocket, we observe that two of the discovered EI ligands act as competitive inhibitors while the third ligand acts as a mixed inhibitor. Characterization of the EI-inhibitor complexes by NMR and Molecular Dynamics simulations reveals key interactions that perturb the fold of the active site and provides structural foundation for the different inhibitory activity of the identified molecular fragments. In particular, we show that contacts between the inhibitor and the side-chain of V292 are crucial to destabilize binding of the substrate to EI. In contrast, mixed inhibition is caused by additional contacts between the inhibitor and ⍺-helix 2 that perturb the active site structure and turnover in an allosteric manner. We expect our results to provide the basis for the development of second generation allosteric inhibitors of increased potency and to suggest novel molecular strategies to combat drug-resistant infections. |
| first_indexed | 2024-12-20T09:55:12Z |
| format | Article |
| id | doaj.art-eea6b0f5ca6e4ad4b37ba133962800b1 |
| institution | Directory Open Access Journal |
| issn | 2590-1524 |
| language | English |
| last_indexed | 2024-12-20T09:55:12Z |
| publishDate | 2020-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Structural Biology: X |
| spelling | doaj.art-eea6b0f5ca6e4ad4b37ba133962800b12022-12-21T19:44:29ZengElsevierJournal of Structural Biology: X2590-15242020-01-014100034An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screeningTrang T. Nguyen0Vincenzo Venditti1Department of Chemistry, Iowa State University, Ames, IA 50011, USADepartment of Chemistry, Iowa State University, Ames, IA 50011, USA; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA; Corresponding author at: Department of Chemistry, Iowa State University, Hach Hall, 2438 Pammel Drive, Ames, IA 50011, USA.Enzyme I (EI), which is the key enzyme to activate the bacterial phosphotransferase system, plays an important role in the regulation of several metabolic pathways and controls the biology of bacterial cells at multiple levels. The conservation and ubiquity of EI among different types of bacteria makes the enzyme a potential target for antimicrobial research. Here, we use NMR-based fragment screening to identify novel inhibitors of EI. We identify three molecular fragments that allosterically inhibit the phosphoryl transfer reaction catalyzed by EI by interacting with the enzyme at a surface pocket located more than 10 Å away from the substrate binding site. Interestingly, although the three molecules share the same binding pocket, we observe that two of the discovered EI ligands act as competitive inhibitors while the third ligand acts as a mixed inhibitor. Characterization of the EI-inhibitor complexes by NMR and Molecular Dynamics simulations reveals key interactions that perturb the fold of the active site and provides structural foundation for the different inhibitory activity of the identified molecular fragments. In particular, we show that contacts between the inhibitor and the side-chain of V292 are crucial to destabilize binding of the substrate to EI. In contrast, mixed inhibition is caused by additional contacts between the inhibitor and ⍺-helix 2 that perturb the active site structure and turnover in an allosteric manner. We expect our results to provide the basis for the development of second generation allosteric inhibitors of increased potency and to suggest novel molecular strategies to combat drug-resistant infections.http://www.sciencedirect.com/science/article/pii/S2590152420300167Mixed inhibitionCompetitive inhibitionAntimicrobial compoundsPrincipal component analysisBacterial phosphotransferase systemTIM barrel enzyme |
| spellingShingle | Trang T. Nguyen Vincenzo Venditti An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening Journal of Structural Biology: X Mixed inhibition Competitive inhibition Antimicrobial compounds Principal component analysis Bacterial phosphotransferase system TIM barrel enzyme |
| title | An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening |
| title_full | An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening |
| title_fullStr | An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening |
| title_full_unstemmed | An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening |
| title_short | An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening |
| title_sort | allosteric pocket for inhibition of bacterial enzyme i identified by nmr based fragment screening |
| topic | Mixed inhibition Competitive inhibition Antimicrobial compounds Principal component analysis Bacterial phosphotransferase system TIM barrel enzyme |
| url | http://www.sciencedirect.com/science/article/pii/S2590152420300167 |
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