Opening of a Cryptic Pocket in β-lactamase Increases Penicillinase Activity
| Autor: | Shreya Raavicharla, Gregory R. Bowman, Upasana L. Mallimadugula, Catherine R. Knoverek, Lewis E. Kay, Thomas E. Frederick, Tairan Yuwen, Enrico Rennella, Sukrit Singh |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Protein Conformation
Open population Stereochemistry cryptic pockets Protein design Molecular Dynamics Simulation 010402 general chemistry 01 natural sciences Omega beta-Lactamases 03 medical and health sciences Molecular dynamics Escherichia coli protein evolution Beta (finance) 030304 developmental biology 0303 health sciences Binding Sites Multidisciplinary Chemistry Drug discovery Escherichia coli Proteins Protein dynamics Proteins Penicillin G Biological Sciences Penicillinase 0104 chemical sciences Biophysics and Computational Biology Saturation transfer Excited state protein dynamics Mutation Biophysics Penicillinase activity Function (biology) |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| DOI: | 10.1101/2021.04.14.439842 |
| Popis: | Significance A protein is a shape-shifter, but it is currently unclear which of the many structures a protein can adopt are relevant for its function. Here, we examine conformations that contain a “cryptic” pocket (i.e., a pocket absent in ligand-free structures). Cryptic pockets have potential utility in drug discovery efforts because they provide a means to target “undruggable” proteins (i.e., proteins lacking known pockets) or enhance rather than inhibit protein function. In this study, we use a combination of thiol-labeling and kinetic assays, NMR, and molecular dynamic simulations to identify the function of the Ω-loop cryptic pocket in β-lactamase enzymes. We find that an open pocket population is beneficial for hydrolysis of the substrate benzylpenicillin. 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|