Structures of the essential efflux pump EfpA from Mycobacterium tuberculosis reveal the mechanisms of substrate transport and small-molecule inhibition

Shuhui Wang; Kun Wang; Kangkang Song; Pengfei Li; Dongying Li; Yajie Sun; Ye Mei; Chen Xu; Maofu Liao

doi:10.21203/rs.3.rs-3740027/v1

Structures of the essential efflux pump EfpA from Mycobacterium tuberculosis reveal the mechanisms of substrate transport and small-molecule inhibition

Res Sq [Preprint]. 2024 Jan 5:rs.3.rs-3740027. doi: 10.21203/rs.3.rs-3740027/v1.

Authors

Shuhui Wang^{1

2}, Kun Wang^{1

3}, Kangkang Song^{4

5}, Pengfei Li⁶, Dongying Li^{1

7}, Yajie Sun⁸, Ye Mei⁹, Chen Xu^{4

5}, Maofu Liao^{1

10

11}

Affiliations

¹ Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
² Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, USA.
³ Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA.
⁴ Department of Biochemistry & Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
⁵ Cryo-EM Core Facility, University of Massachusetts Medical School, Worcester, MA, USA.
⁶ Single Particle, LLC, San Diego, CA, USA.
⁷ Present address: Cryo-electron microscopy center, Southern University of Science and Technology, Shenzhen, China.
⁸ Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
⁹ State Key Laboratory of Precision Spectroscopy, School of Physics and Electronic Science, East China Normal University, Shanghai, China.
¹⁰ Present address: Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, China.
¹¹ Present address: Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China.

Abstract

As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA are poorly understood. Herein we report cryo-EM structures of EfpA in outward-open conformation, either bound to three endogenous lipids or the inhibitor BRD-8000.3. Three lipids inside EfpA span from the inner leaflet to the outer leaflet of the membrane. BRD-8000.3 occupies one lipid site at the level of inner membrane leaflet, competitively inhibiting lipid binding. EfpA resembles the related lysophospholipid transporter MFSD2A in both overall structure and lipid binding sites, and may function as a lipid flippase. Combining AlphaFold-predicted EfpA structure, which is inward-open, we propose a complete conformational transition cycle for EfpA. Together, our results provide a structural and mechanistic foundation to comprehend EfpA function and develop EfpA-targeting anti-TB drugs.

Publication types

Preprint

Grants and funding

U24 GM129547/GM/NIGMS NIH HHS/United States