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Opening of a Cryptic Pocket in β-lactamase Increases Penicillinase Activity

View ORCID ProfileCatherine R Knoverek, Upasana L Mallimadugula, Sukrit Singh, Enrico Rennella, Thomas E Frederick, Tairan Yuwen, Shreya Raavicharla, View ORCID ProfileLewis E Kay, View ORCID ProfileGregory R Bowman
doi: https://doi.org/10.1101/2021.04.14.439842
Catherine R Knoverek
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, United States
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Upasana L Mallimadugula
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, United States
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Sukrit Singh
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, United States
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Enrico Rennella
2Department of Molecular Genetics, The University of Toronto, Toronto, Canada
3Department of Biochemistry, The University of Toronto, Toronto, Canada
4Department of Chemistry, The University of Toronto, Toronto, Canada
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Thomas E Frederick
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, United States
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Tairan Yuwen
5Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Peking University, Beijing, China
6State Key Laboratory of Nature and Biomimetic Drugs, Peking University, Beijing, China
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Shreya Raavicharla
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, United States
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Lewis E Kay
2Department of Molecular Genetics, The University of Toronto, Toronto, Canada
3Department of Biochemistry, The University of Toronto, Toronto, Canada
4Department of Chemistry, The University of Toronto, Toronto, Canada
7Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Canada
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Gregory R Bowman
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, United States
8Center for the Science and Engineering of Living Systems, Washington University in St. Louis, St. Louis, United States
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  • For correspondence: g.bowman@wustl.edu
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Abstract

Understanding the functional role of protein excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in β-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across β-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM β-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR CEST experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket’s functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 14, 2021.
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Opening of a Cryptic Pocket in β-lactamase Increases Penicillinase Activity
Catherine R Knoverek, Upasana L Mallimadugula, Sukrit Singh, Enrico Rennella, Thomas E Frederick, Tairan Yuwen, Shreya Raavicharla, Lewis E Kay, Gregory R Bowman
bioRxiv 2021.04.14.439842; doi: https://doi.org/10.1101/2021.04.14.439842
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Opening of a Cryptic Pocket in β-lactamase Increases Penicillinase Activity
Catherine R Knoverek, Upasana L Mallimadugula, Sukrit Singh, Enrico Rennella, Thomas E Frederick, Tairan Yuwen, Shreya Raavicharla, Lewis E Kay, Gregory R Bowman
bioRxiv 2021.04.14.439842; doi: https://doi.org/10.1101/2021.04.14.439842

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