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

Research output: Contribution to journalArticlepeer-review

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 chemical exchange saturation transfer 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.

Original languageEnglish
Article numbere2106473118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number47
DOIs
StatePublished - Nov 23 2021

Keywords

  • Cryptic pockets
  • Protein dynamics
  • Protein evolution

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