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bioRxiv · 10.1101/2024.02.22.581608

The assembly and efflux processes revealed by in situ structures of the ABC-type tripartite pump MacAB-TolC

Huo, T.·Yu, Z.·Cao, J.-L.·Shi, X.·wang, z.
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Abstract

The MacAB-TolC tripartite efflux pump is widely distributed in Gram-negative bacteria, extruding antibiotics and virulence factors that lead to multidrug resistance and pathogenicity. This pump spans the cell envelope through its three components, an inner membrane ATP-binding cassette (ABC) transporter MacB, a periplasmic adaptor protein MacA, and an outer membrane protein TolC. However, the assembly process and efflux mechanism of the MacAB-TolC in cells remain unclear. Here, we resolve the in situ structures of Escherichia coli (E. coli) MacAB-TolC efflux pump by electron cryo-tomography and subtomogram averaging. In E. coli without antibiotic treatment, we observe a fully assembled MacAB-TolC pump with a weak constriction density in the middle of the MacA region. When E. coli cells were treated with erythromycin, we discovered the emergence of MacA-TolC subcomplexes, indicating flexible binding of MacB in the presence of an antibiotic substrate. This finding was further validated by in vivo crosslinking results. Together, our data present the in situ assembly process of the MacAB-TolC and derive a substrate-driven working model for ABC-type tripartite pump.

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Huo, T., Yu, Z., Cao, J.-L., Shi, X., wang, z.. 2024-02-22. The assembly and efflux processes revealed by in situ structures of the ABC-type tripartite pump MacAB-TolC. https://doi.org/10.1101/2024.02.22.581608

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