Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

Title: Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance

Abstract

CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restrictingmore » the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.« less

Authors:
; ; ; ; ; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1770499
Alternate Identifier(s):
OSTI ID: 1760246
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Published Article
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Name: Journal of Biological Chemistry Journal Volume: 295 Journal Issue: 21; Journal ID: ISSN 0021-9258
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; antibiotic resistance; enzyme kinetics; enzyme mutation; enzyme structure; protein conformation; protein crystallization; conformational change; protein stability; protein structure; protein drug interaction; beta-lactam antibiotic; beta-lactamase; cephalosporin

Citation Formats

Brown, Cameron A., Hu, Liya, Sun, Zhizeng, Patel, Meha P., Singh, Sukrit, Porter, Justin R., Sankaran, Banumathi, Prasad, B. V. Venkataram, Bowman, Gregory R., and Palzkill, Timothy. Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance. United States: N. p., 2020. Web. doi:10.1074/jbc.RA119.012489.
Copy to clipboard
Brown, Cameron A., Hu, Liya, Sun, Zhizeng, Patel, Meha P., Singh, Sukrit, Porter, Justin R., Sankaran, Banumathi, Prasad, B. V. Venkataram, Bowman, Gregory R., & Palzkill, Timothy. Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance. United States. https://doi.org/10.1074/jbc.RA119.012489
Copy to clipboard
Brown, Cameron A., Hu, Liya, Sun, Zhizeng, Patel, Meha P., Singh, Sukrit, Porter, Justin R., Sankaran, Banumathi, Prasad, B. V. Venkataram, Bowman, Gregory R., and Palzkill, Timothy. Fri . "Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance". United States. https://doi.org/10.1074/jbc.RA119.012489.
Copy to clipboard
@article{osti_1770499,
title = {Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance},
author = {Brown, Cameron A. and Hu, Liya and Sun, Zhizeng and Patel, Meha P. and Singh, Sukrit and Porter, Justin R. and Sankaran, Banumathi and Prasad, B. V. Venkataram and Bowman, Gregory R. and Palzkill, Timothy},
abstractNote = {CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.},
doi = {10.1074/jbc.RA119.012489},
journal = {Journal of Biological Chemistry},
number = 21,
volume = 295,
place = {United States},
year = {Fri May 01 00:00:00 EDT 2020},
month = {Fri May 01 00:00:00 EDT 2020}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1074/jbc.RA119.012489
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

The β-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter
journal, September 2006

Biochemical characterisation of the CTX-M-14 β-lactamase
journal, February 2007

Additivity of mutational effects in proteins
journal, September 1990

Atomic Resolution Structures of CTX-M β-Lactamases: Extended Spectrum Activities from Increased Mobility and Decreased Stability
journal, April 2005

Darwinian Evolution Can Follow Only Very Few Mutational Paths to Fitter Proteins
journal, April 2006

Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes
journal, May 1986

The Flexibility of a Distant Loop Modulates Active Site Motion and Product Release in Ribonuclease A
journal, August 2009

  • Doucet, Nicolas; Watt, Eric D.; Loria, J. Patrick
  • Biochemistry, Vol. 48, Issue 30
  • DOI: 10.1021/bi900830g

Molecular Basis for the Catalytic Specificity of the CTX-M Extended-Spectrum β-Lactamases
journal, December 2014

  • Adamski, Carolyn J.; Cardenas, Ana Maria; Brown, Nicholas G.
  • Biochemistry, Vol. 54, Issue 2
  • DOI: 10.1021/bi501195g

Enzyme catalysis: not different, just better
journal, March 1991

Negative Epistasis and Evolvability in TEM-1 β-Lactamase—The Thin Line between an Enzyme's Conformational Freedom and Disorder
journal, July 2015

  • Dellus-Gur, Eynat; Elias, Mikael; Caselli, Emilia
  • Journal of Molecular Biology, Vol. 427, Issue 14
  • DOI: 10.1016/j.jmb.2015.05.011

GROMACS: Fast, flexible, and free
journal, January 2005

  • Van Der Spoel, David; Lindahl, Erik; Hess, Berk
  • Journal of Computational Chemistry, Vol. 26, Issue 16
  • DOI: 10.1002/jcc.20291

Automatic atom type and bond type perception in molecular mechanical calculations
journal, October 2006

  • Wang, Junmei; Wang, Wei; Kollman, Peter A.
  • Journal of Molecular Graphics and Modelling, Vol. 25, Issue 2
  • DOI: 10.1016/j.jmgm.2005.12.005

Three Decades of the Class A β-Lactamase Acyl-Enzyme
journal, October 2009

Comparison of simple potential functions for simulating liquid water
journal, July 1983

  • Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D.
  • The Journal of Chemical Physics, Vol. 79, Issue 2
  • DOI: 10.1063/1.445869

GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers
journal, September 2015

Epistasis as the primary factor in molecular evolution
journal, October 2012

  • Breen, Michael S.; Kemena, Carsten; Vlasov, Peter K.
  • Nature, Vol. 490, Issue 7421
  • DOI: 10.1038/nature11510

Bacterial Resistance to β-Lactam Antibiotics:  Compelling Opportunism, Compelling Opportunity †
journal, February 2005

  • Fisher, Jed F.; Meroueh, Samy O.; Mobashery, Shahriar
  • Chemical Reviews, Vol. 105, Issue 2
  • DOI: 10.1021/cr030102i

Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases
journal, August 2015

  • Patel, Meha P.; Fryszczyn, Bartlomiej G.; Palzkill, Timothy
  • Antimicrobial Agents and Chemotherapy, Vol. 59, Issue 11
  • DOI: 10.1128/AAC.00618-15

Development and testing of a general amber force field
journal, January 2004

  • Wang, Junmei; Wolf, Romain M.; Caldwell, James W.
  • Journal of Computational Chemistry, Vol. 25, Issue 9
  • DOI: 10.1002/jcc.20035

Protein Dynamism and Evolvability
journal, April 2009

Effect of D240G substitution in a novel ESBL CTX-M-27
journal, June 2003

  • Bonnet, R.
  • Journal of Antimicrobial Chemotherapy, Vol. 52, Issue 1
  • DOI: 10.1093/jac/dkg256

ACPYPE - AnteChamber PYthon Parser interfacE
journal, January 2012

Synergistic effects of functionally distinct substitutions in β-lactamase variants shed light on the evolution of bacterial drug resistance
journal, October 2018

  • Patel, Meha P.; Hu, Liya; Brown, Cameron A.
  • Journal of Biological Chemistry, Vol. 293, Issue 46
  • DOI: 10.1074/jbc.RA118.003792

Conformational dynamics and enzyme evolution
journal, July 2018

  • Petrović, Dušan; Risso, Valeria A.; Kamerlin, Shina Caroline Lynn
  • Journal of The Royal Society Interface, Vol. 15, Issue 144
  • DOI: 10.1098/rsif.2018.0330

Epidemiological Expansion, Structural Studies, and Clinical Challenges of New β-Lactamases from Gram-Negative Bacteria
journal, October 2011

Cooperative Changes in Solvent Exposure Identify Cryptic Pockets, Switches, and Allosteric Coupling
journal, March 2019

  • Porter, Justin R.; Moeder, Katelyn E.; Sibbald, Carrie A.
  • Biophysical Journal, Vol. 116, Issue 5
  • DOI: 10.1016/j.bpj.2018.11.3144

β-Lactamases: protein evolution in real time
journal, August 1998

Conformational diversity and protein evolution – a 60-year-old hypothesis revisited
journal, July 2003

CTX-M Enzymes: Origin and Diffusion
journal, January 2012

  • Cantón, Rafael; González-Alba, José María; Galán, Juan Carlos
  • Frontiers in Microbiology, Vol. 3
  • DOI: 10.3389/fmicb.2012.00110

Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli
journal, November 1983

GROMACS: A message-passing parallel molecular dynamics implementation
journal, September 1995

  • Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R.
  • Computer Physics Communications, Vol. 91, Issue 1-3
  • DOI: 10.1016/0010-4655(95)00042-E

Role of a Mutation at Position 167 of CTX-M-19 in Ceftazidime Hydrolysis
journal, May 2004

Contributions of Aspartate 49 and Phenylalanine 142 Residues of a Tight Binding Inhibitory Protein of β-Lactamases
journal, January 1999

  • Petrosino, Joseph; Rudgers, Gary; Gilbert, Hiram
  • Journal of Biological Chemistry, Vol. 274, Issue 4
  • DOI: 10.1074/jbc.274.4.2394

Growing Group of Extended-Spectrum  -Lactamases: the CTX-M Enzymes
journal, December 2003

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

The Acylation Mechanism of CTX-M β-Lactamase at 0.88 Å Resolution
journal, May 2007

  • Chen, Yu; Bonnet, Richard; Shoichet, Brian K.
  • Journal of the American Chemical Society, Vol. 129, Issue 17
  • DOI: 10.1021/ja0712064

Molecular structure of the acyl-enzyme intermediate in β-lactam hydrolysis at 1.7 Å resolution
journal, October 1992

  • Strynadka, Natalie C. J.; Adachi, Hiroyuki; Jensen, Susan E.
  • Nature, Vol. 359, Issue 6397
  • DOI: 10.1038/359700a0

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Natural evolution of TEM-1 β-lactamase: experimental reconstruction and clinical relevance
journal, November 2010

High-Level Resistance to Ceftazidime Conferred by a Novel Enzyme, CTX-M-32, Derived from CTX-M-1 through a Single Asp240-Gly Substitution
journal, June 2004

A standard numbering scheme for the class A β-lactamases
journal, May 1991

  • Ambler, R. P.; Coulson, A. F. W.; Frère, J. M.
  • Biochemical Journal, Vol. 276, Issue 1
  • DOI: 10.1042/bj2760269

Prediction of protein B-factor profiles
journal, January 2005

  • Yuan, Zheng; Bailey, Timothy L.; Teasdale, Rohan D.
  • Proteins: Structure, Function, and Bioinformatics, Vol. 58, Issue 4
  • DOI: 10.1002/prot.20375

CTX-M-type β-lactamases: A successful story of antibiotic resistance
journal, August 2013

  • D’Andrea, Marco Maria; Arena, Fabio; Pallecchi, Lucia
  • International Journal of Medical Microbiology, Vol. 303, Issue 6-7
  • DOI: 10.1016/j.ijmm.2013.02.008

What Makes a Protein Fold Amenable to Functional Innovation? Fold Polarity and Stability Trade-offs
journal, July 2013

  • Dellus-Gur, Eynat; Toth-Petroczy, Agnes; Elias, Mikael
  • Journal of Molecular Biology, Vol. 425, Issue 14
  • DOI: 10.1016/j.jmb.2013.03.033

Modelling proteins’ hidden conformations to predict antibiotic resistance
journal, October 2016

  • Hart, Kathryn M.; Ho, Chris M. W.; Dutta, Supratik
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms12965

Empirical fitness landscapes and the predictability of evolution
journal, June 2014

  • de Visser, J. Arjan G. M.; Krug, Joachim
  • Nature Reviews Genetics, Vol. 15, Issue 7
  • DOI: 10.1038/nrg3744

A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations
journal, October 2003

  • Duan, Yong; Wu, Chun; Chowdhury, Shibasish
  • Journal of Computational Chemistry, Vol. 24, Issue 16
  • DOI: 10.1002/jcc.10349

Multiple Global Suppressors of Protein Stability Defects Facilitate the Evolution of Extended-Spectrum TEM β-Lactamases
journal, December 2010

  • Brown, Nicholas G.; Pennington, Jeanine M.; Huang, Wanzhi
  • Journal of Molecular Biology, Vol. 404, Issue 5
  • DOI: 10.1016/j.jmb.2010.10.008

Mutational Events in Cefotaximase Extended-Spectrum β-Lactamases of the CTX-M-1 Cluster Involved in Ceftazidime Resistance
journal, April 2008

  • Novais, Ângela; Cantón, Rafael; Coque, Teresa M.
  • Antimicrobial Agents and Chemotherapy, Vol. 52, Issue 7
  • DOI: 10.1128/AAC.01658-07

Conformational Motions Regulate Phosphoryl Transfer in Related Protein Tyrosine Phosphatases
journal, August 2013

A natural polymorphism in  -lactamase is a global suppressor
journal, August 1997

  • Huang, W.; Palzkill, T.
  • Proceedings of the National Academy of Sciences, Vol. 94, Issue 16
  • DOI: 10.1073/pnas.94.16.8801

Genetic and Structural Characterization of an L201P Global Suppressor Substitution in TEM-1 β-Lactamase
journal, December 2008

  • Marciano, David C.; Pennington, Jeanine M.; Wang, Xiaohu
  • Journal of Molecular Biology, Vol. 384, Issue 1
  • DOI: 10.1016/j.jmb.2008.09.009

Correlation of Levels of Folded Recombinant p53 in Escherichia coli with Thermodynamic Stability in Vitro
journal, September 2007

  • Mayer, Sebastian; Rüdiger, Stefan; Ang, Hwee Ching
  • Journal of Molecular Biology, Vol. 372, Issue 1
  • DOI: 10.1016/j.jmb.2007.06.044

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis
journal, February 2015

  • Stojanoski, Vlatko; Chow, Dar-Chone; Hu, Liya
  • Journal of Biological Chemistry, Vol. 290, Issue 16
  • DOI: 10.1074/jbc.M114.633438

OXA-48-Mediated Ceftazidime-Avibactam Resistance Is Associated with Evolutionary Trade-Offs
journal, March 2019

    Sort by:
    [ × clear filter / sort ]

    Similar Records in DOE PAGES and OSTI.GOV collections: