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Reconstitution of RND tripartite multidrug complexes for single-particle electron microscopy
Esther Boyer1, Laetitia Daury1, Marie-France Giraud1
1Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR5248, Pessac, France.
Abstract:
Tripartite multidrug RND efflux pumps (TEP) made of an inner membrane RND transporter, an outer membrane factor (OMF) and a periplasmic adaptor protein (PAP) form a canal for expelling drugs through the cell wall of Gram-negative bacteria. Various RND transporters function with their cognate PAP and OMF although the rule is not so strict and an exchange with other OMF or PAP is not detrimental to TEP function. The molecular mechanism by which OMF-PAP-RND tripartite complexes are assembled is still poorly understood, as is the rule governing partner selection, leading to exchanges of TEP partners. Here, we present the reconstitution of the Pseudomonas aeruginosa OprM-MexA-MexB TEP and the chimeric OprN-MexA-MexB TEP into nanodiscs, combined with structural characterization by single-particle electron microscopy. This method allows assessment of the ability to form TEPs with cognate and non-cognate partners under controlled conditions, providing insight into the molecular recognition of the TEP components. The reconstitutions provide a better understanding of the effect of pH and other factors on TEP assembly.
Insights
Tripartite multidrug efflux pumps (TEPs) in Gram-negative bacteria assemble using inner membrane transporters, outer membrane factors, and periplasmic adaptor proteins. This study reveals molecular recognition rules governing TEP component assembly and function.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Gram-negative bacteria utilize tripartite efflux pumps (TEPs) for multidrug resistance.
- TEPs comprise an RND transporter, an outer membrane factor (OMF), and a periplasmic adaptor protein (PAP).
- The assembly mechanism and partner selection rules for TEPs remain poorly understood.
Purpose of the Study:
- To investigate the molecular recognition and assembly of TEP components.
- To understand the rules governing partner selection in TEP formation.
- To elucidate the impact of factors like pH on TEP assembly.
Main Methods:
- Reconstitution of Pseudomonas aeruginosa OprM-MexA-MexB TEP and chimeric OprN-MexA-MexB TEP in nanodiscs.
- Structural characterization using single-particle electron microscopy.
- Assessment of TEP formation with cognate and non-cognate partners.
Main Results:
- Successful reconstitution of TEPs in nanodiscs allowed controlled assessment of component interactions.
- Insights into the molecular recognition governing TEP assembly were obtained.
- The influence of pH and other factors on TEP assembly was elucidated.
Conclusions:
- The study provides a method to assess TEP formation and component interactions.
- Understanding TEP assembly mechanisms can inform strategies to combat multidrug resistance in bacteria.
- This work sheds light on the flexibility and specificity of TEP component interactions.
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