TY - JOUR
T1 - Opening of a cryptic pocket in β-lactamase increases penicillinase activity
AU - Knoverek, Catherine R.
AU - Mallimadugula, Upasana L.
AU - Singh, Sukrit
AU - Rennella, Enrico
AU - Frederick, Thomas E.
AU - Yuwen, Tairan
AU - Raavicharla, Shreya
AU - Kay, Lewis E.
AU - Bowman, Gregory R.
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank the community scientists of Folding@home for donating their computing resources. This work was funded by NIH Grant R01GM12400701 (G.R.B.), NSF CAREER Award MCB-1552471 (G.R.B.), Canadian Institutes of Health Research FDN-503573 (L.E.K.), and the Natural Sciences and Engineering Research Council of Canada (L.E.K.). G.R.B. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund and a Packard Fellowship for Science and Engineering from the David and Lucile Packard Foundation. L.E.K. holds a Canada Research Chair in Biochemistry.
Publisher Copyright:
© 2021 National Academy of Sciences. All rights reserved.
PY - 2021/11/23
Y1 - 2021/11/23
N2 - 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.
AB - 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.
KW - Cryptic pockets
KW - Protein dynamics
KW - Protein evolution
UR - http://www.scopus.com/inward/record.url?scp=85120317654&partnerID=8YFLogxK
U2 - 10.1073/pnas.2106473118
DO - 10.1073/pnas.2106473118
M3 - Article
C2 - 34799442
AN - SCOPUS:85120317654
VL - 118
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 47
M1 - e2106473118
ER -