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Updated: May 22, 2026
Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis
Published on: October 28, 2016
Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis
Yann Ferrandez1, Laura Monlezun, Gilles Phan
1Laboratoire de Cristallographie et RMN Biologiques, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Paris, France.
Abstract:
Multidrug resistance has become a serious concern in the treatment of bacterial infections. A prominent role is ascribed to the active efflux of xenobiotics out of the bacteria by a tripartite protein machinery. The mechanism of drug extrusion is rather well understood, thanks to the X-ray structures obtained for the Escherichia coli TolC/AcrA/AcrB model system and the related Pseudomonas aeruginosa OprM/MexA/MexB. However, many questions remain unresolved, in particular the stoichiometry of the efflux pump assembly. On the basis of blue native polyacrylamide gel electrophoresis (BN-PAGE) (Wittig et al., Nat. Protoc. 2006, 1, 418-428), we analyzed the binding stoichiometry of both palmitylated and non-palmitylated MexA with the cognate partner OprM trimer at different ratios and detergent conditions. We found that β-octyl glucopyranoside (β-OG) detergent was not suitable for this technique. Then we proved that MexA has to be palmitylated in order to stabilized the complex formation with OprM. Finally, we provided evidence for a two by two (2, 4, 6, or upper) binding of palmitylated MexA per trimer of OprM.
Insights
Multidrug resistance in bacteria is a growing problem. This study reveals that palmitylated MexA binds to the OprM trimer in a 2:2, 4:2, 6:2, or higher ratio, clarifying efflux pump stoichiometry.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Multidrug resistance (MDR) in bacteria poses a significant clinical challenge.
- Active efflux pumps, particularly tripartite systems, are key contributors to MDR.
- Understanding the stoichiometry of these efflux pump assemblies is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the binding stoichiometry of MexA and OprM in Pseudomonas aeruginosa.
- To determine the role of MexA palmitylation in complex formation with OprM.
- To elucidate the precise assembly of these bacterial efflux pumps.
Main Methods:
- Blue native polyacrylamide gel electrophoresis (BN-PAGE) was employed to analyze protein complex formation.
- The binding stoichiometry of palmitylated and non-palmitylated MexA with OprM was assessed under varying conditions.
- The suitability of β-octyl glucopyranoside (β-OG) detergent for BN-PAGE was evaluated.
Main Results:
- β-octyl glucopyranoside (β-OG) detergent was found to be unsuitable for BN-PAGE analysis of the MexA-OprM complex.
- Palmitylation of MexA is essential for stabilizing its complex formation with the OprM trimer.
- Evidence was provided for a 2:2, 4:2, 6:2, or higher binding stoichiometry of palmitylated MexA to the OprM trimer.
Conclusions:
- MexA must be palmitylated to effectively bind and stabilize the OprM trimer.
- The stoichiometry of palmitylated MexA binding to OprM is multi-meric, suggesting a flexible assembly.
- These findings advance our understanding of bacterial efflux pump structure and function, aiding in the fight against multidrug resistance.
