Skip to main content

Optimization of a Noncanonical Anti-infective: Interrogation of the Target Binding Pocket for a Small-Molecule Inhibitor of Escherichia coli Polysaccharide Capsule Expression.

Publication ,  Journal Article
Arshad, M; Beggs, GA; Brennan, RG; Seed, PC
Published in: Antimicrob Agents Chemother
December 16, 2020

We previously identified a small-molecule inhibitor of capsule biogenesis (designated DU011) and identified its target as MprA, a MarR family transcriptional repressor of multidrug efflux pumps. Unlike other proposed MprA ligands, such as salicylate and 2,4-dinitrophenol (DNP), DU011 does not alter Escherichia coli antibiotic resistance and has significantly enhanced inhibition of capsule expression. We hypothesized that the potency and the unique action of DU011 are due to novel interactions with the MprA binding pocket and the conformation assumed by MprA upon binding DU011 relative to other ligands. To understand the dynamics of MprA-DU011 interaction, we performed hydrogen-deuterium exchange mass spectrometry (HDX-MS); this suggested that four peptide regions undergo conformational changes upon binding DU011. We conducted isothermal calorimetric titration (ITC) to quantitatively characterize MprA binding to DU011 and canonical ligands and observed a distinct two-site binding isotherm associated with the binding reaction of MprA to DU011; however, salicylate and DNP showed a one-site binding isotherm with lower affinity. To elucidate the binding pocket(s) of MprA, we selected single point mutants of MprA that included mutated residues predicted to be within the putative binding pocket (Q51A, F58A, and E65D) as well as on or near the DNA-binding domain (L81A, S83T, and T86A). Our ITC studies suggest that two of the tested MprA mutants had lower affinity for DU011: Q51A and F58A. In addition to elucidating the MprA binding pocket for DU011, we studied the binding of these mutants to salicylate and DNP to reveal the binding pockets of these canonical ligands.

Duke Scholars

Published In

Antimicrob Agents Chemother

DOI

EISSN

1098-6596

Publication Date

December 16, 2020

Volume

65

Issue

1

Location

United States

Related Subject Headings

  • Protein Binding
  • Polysaccharides
  • Microbiology
  • Ligands
  • Escherichia coli Proteins
  • Escherichia coli
  • Drug Resistance, Microbial
  • Binding Sites
  • Anti-Bacterial Agents
  • 3214 Pharmacology and pharmaceutical sciences
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Arshad, M., Beggs, G. A., Brennan, R. G., & Seed, P. C. (2020). Optimization of a Noncanonical Anti-infective: Interrogation of the Target Binding Pocket for a Small-Molecule Inhibitor of Escherichia coli Polysaccharide Capsule Expression. Antimicrob Agents Chemother, 65(1). https://doi.org/10.1128/AAC.01208-20
Arshad, Mehreen, Grace A. Beggs, Richard G. Brennan, and Patrick C. Seed. “Optimization of a Noncanonical Anti-infective: Interrogation of the Target Binding Pocket for a Small-Molecule Inhibitor of Escherichia coli Polysaccharide Capsule Expression.Antimicrob Agents Chemother 65, no. 1 (December 16, 2020). https://doi.org/10.1128/AAC.01208-20.
Arshad, Mehreen, et al. “Optimization of a Noncanonical Anti-infective: Interrogation of the Target Binding Pocket for a Small-Molecule Inhibitor of Escherichia coli Polysaccharide Capsule Expression.Antimicrob Agents Chemother, vol. 65, no. 1, Dec. 2020. Pubmed, doi:10.1128/AAC.01208-20.

Published In

Antimicrob Agents Chemother

DOI

EISSN

1098-6596

Publication Date

December 16, 2020

Volume

65

Issue

1

Location

United States

Related Subject Headings

  • Protein Binding
  • Polysaccharides
  • Microbiology
  • Ligands
  • Escherichia coli Proteins
  • Escherichia coli
  • Drug Resistance, Microbial
  • Binding Sites
  • Anti-Bacterial Agents
  • 3214 Pharmacology and pharmaceutical sciences