TY - JOUR
T1 - Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance
AU - Brown, Cameron A.
AU - Hu, Liya
AU - Sun, Zhizeng
AU - Patel, Meha P.
AU - Singh, Sukrit
AU - Porter, Justin R.
AU - Sankaran, Banumathi
AU - Venkataram Prasad, B. V.
AU - Bowman, Gregory R.
AU - Palzkill, Timothy
N1 - Funding Information:
Acknowledgments—We thank Hiram Gilbert for discussions and comments on the manuscript. The ALS-ENABLE Beamlines are supported in part by National Institutes of Health, NIGMS Grant P30 GM124169-01. The Advanced Light Source is a Department of Energy Office of Science User Facility under Contract DE-AC02-05CH11231.
Funding Information:
This work was supported by National Institutes of Health Grants R01 AI32956 (to T. P.) and R01 GM12400701 (to G. R. B.) and by Robert Welch Foundation Grant Q1279 (to B. V. V. P.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. thank Hiram Gilbert for discussions and comments on the manuscript. The ALS-ENABLE Beamlines are supported in part by National Institutes of Health, NIGMS Grant P30 GM124169-01. The Advanced Light Source is a Department of Energy Office of Science User Facility under Contract DE-AC02-05CH11231.
Funding Information:
1 Supported by National Institutes of Health Training Grant T32 GM120011.
Funding Information:
This work was supported by National Institutes of Health Grants R01 AI32956 (to T. P.) and R01 GM12400701 (to G. R. B.) and by Robert Welch Founda-tion Grant Q1279 (to B. V. V. P.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.
PY - 2020/5/22
Y1 - 2020/5/22
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85085244795&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA119.012489
DO - 10.1074/jbc.RA119.012489
M3 - Article
C2 - 32299911
AN - SCOPUS:85085244795
VL - 295
SP - 7376
EP - 7390
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 21
ER -