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Title: Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

Abstract

Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic‐resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin‐binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor‐free form and in complex with penicillin V are reported. We used site‐directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of β‐lactams. Structural comparison of the PonA1 apo‐form and the antibiotic‐bound form shows that binding of penicillin V induces conformational changes in the position of the loop β4′‐α3 surrounding the penicillin‐binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested β‐lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerancemore » to specific cell wall‐targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections. Database Structural data are available in the PDB database under the accession numbers 5CRF and 5CXW .« less

Authors:
 [1];  [2];  [3];  [4];  [1];  [5];  [1]
+ Show Author Affiliations
  1. Department of Biochemistry and Molecular Genetics Feinberg School of Medicine Northwestern University Chicago IL USA, Midwest Center for Structural Genomics (MCSG) Biosciences Division Argonne National Laboratory Argonne, IL USA, Center for Structural Genomics of Infectious Diseases (CSGID) Feinberg School of Medicine Northwestern University Chicago, IL USA
  2. Department of Immunology and Infectious Disease Harvard T.H. Chan School of Public Health Boston MA USA
  3. Midwest Center for Structural Genomics (MCSG) Biosciences Division Argonne National Laboratory Argonne, IL USA, High Throughput Analysis Laboratory and Department of Molecular Biosciences Northwestern University Evanston IL USA
  4. Life Science Collaborative Access Team Synchrotron Research Center Northwestern University Evanston IL USA
  5. Department of Immunology and Infectious Disease Harvard T.H. Chan School of Public Health Boston MA USA, Department of Microbiology and Immunobiology Harvard Medical School Boston MA USA
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1254659
Alternate Identifier(s):
OSTI ID: 1254660; OSTI ID: 1625876
Grant/Contract Number:  
AC02-06CH11357; [085P1000817]; NIH [GM074942]; NSF [DGE1144152; DGE0946799; MCB 1024945; HHS [HHSN272200700058C; HHSN2722012 00026C]
Resource Type:
Published Article
Journal Name:
Federation of European Biochemical Societies (FEBS) Journal
Additional Journal Information:
Journal Name: Federation of European Biochemical Societies (FEBS) Journal Journal Volume: 283 Journal Issue: 12; Journal ID: ISSN 1742-464X
Publisher:
Wiley-Blackwell
Country of Publication:
United Kingdom
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; Biochemistry & Molecular Biology; b-lactams; antibiotic resistance; PBP; PonA1; transpeptidase domain; tuberculosis

Citation Formats

Filippova, Ekaterina V., Kieser, Karen J., Luan, Chi‐Hao, Wawrzak, Zdzislaw, Kiryukhina, Olga, Rubin, Eric J., and Anderson, Wayne F. Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance. United Kingdom: N. p., 2016. Web. doi:10.1111/febs.13738.
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Filippova, Ekaterina V., Kieser, Karen J., Luan, Chi‐Hao, Wawrzak, Zdzislaw, Kiryukhina, Olga, Rubin, Eric J., & Anderson, Wayne F. Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance. United Kingdom. https://doi.org/10.1111/febs.13738
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Filippova, Ekaterina V., Kieser, Karen J., Luan, Chi‐Hao, Wawrzak, Zdzislaw, Kiryukhina, Olga, Rubin, Eric J., and Anderson, Wayne F. Wed . "Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance". United Kingdom. https://doi.org/10.1111/febs.13738.
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@article{osti_1254659,
title = {Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance},
author = {Filippova, Ekaterina V. and Kieser, Karen J. and Luan, Chi‐Hao and Wawrzak, Zdzislaw and Kiryukhina, Olga and Rubin, Eric J. and Anderson, Wayne F.},
abstractNote = {Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic‐resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin‐binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor‐free form and in complex with penicillin V are reported. We used site‐directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of β‐lactams. Structural comparison of the PonA1 apo‐form and the antibiotic‐bound form shows that binding of penicillin V induces conformational changes in the position of the loop β4′‐α3 surrounding the penicillin‐binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested β‐lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall‐targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections. Database Structural data are available in the PDB database under the accession numbers 5CRF and 5CXW .},
doi = {10.1111/febs.13738},
journal = {Federation of European Biochemical Societies (FEBS) Journal},
number = 12,
volume = 283,
place = {United Kingdom},
year = {Wed May 25 00:00:00 EDT 2016},
month = {Wed May 25 00:00:00 EDT 2016}
}
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Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1111/febs.13738
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Cited by: 17 works
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Figures / Tables:

Fig. 1 Fig. 1: Structure of the transpeptidase domain of PonA1. (A) Ribbon diagram of the C-terminal transpeptidase domain of PonA1 monomer from Mycobacterium tuberculosis. The small adjacent N-terminal segment of the PonA1 transpeptidase domain is colored in blue and cyan. The PonA1 penicillin-binding domain is colored in green. Residues of themore » hydrophobic C-terminal segment and cysteine residues are shown as a ball-and-stick model, where carbon atoms are in green (or blue for cysteines), nitrogen atoms in blue, oxygen atoms in red, and sulfur atoms in pink. Residues composing conserved motifs in the penicillin-binding pocket of PBPs are shown as a cylinder model in crimson. The secondary structure elements are labeled. (B) Electrostatic surface representation. The surface is colored by surface potential charge from negative in red to positive in blue. (C) Comparison of PonA1 structures with (gold) and without penicillin V (green). Penicillin V is shown as a cylinder model in dark purple. The disposition of the loops in superimposed structures is marked with arrows.« less
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