MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

Abstract

The cell envelope of Mycobacterium tuberculosis is notable for the abundance of mycolic acids (MAs), which are essential to mycobacterial viability, and other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, contributes to virulence and antibiotic resistance. Yet, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), but the exact function of MmpL3 remains elusive. Here, we report a high-resolution crystal structure of M. smegmatis MmpL3, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.

Cryo-EM structure and resistance landscape of M. tuberculosis MmpL3: An emergent therapeutic target

“…However, cryo-EM imaging of the protein purified in lauryl maltose neopentyl glycol (LMNG) permitted its three-dimensional reconstruction to a resolution of 3.0 Å (Figures S1B-S1E). Our map reveals MmpL3 1-753 to be monomeric (Figure 1A), consistent with the Msmg homolog (Su et al, 2019;Zhang et al, 2019) (Figure S2A). The cryo-EM map was of sufficient quality to confidently model almost the entirety (residues 1-342, 378-752) of the construct (Figures 1B and S1F; Table S1).…”

“…Similar to structures of the Msmg MmpL3 homolog (Su et al, 2019;Zhang et al, 2019), the Mtb protein has an extensive cavity enclosed by the PD (Figure 1C). The cavity comprises a central vestibule between the PN and PC lobes, out of which branch two descending channels, each constricted in the vicinity of the PD-TMD ''neck.''…”

“…A key question in the mechanism of MmpL3 is how this protein handles the large and complex TMM lipid. The previous Msmg MmpL3 structures were co-crystallized with three unique PD occupants (Figure 2B); pairs of the detergents lauryl-6-trehaloside (6DDTre, a structural analog of TMM) and dodecyl maltoside (DDM) (Zhang et al, 2019), as well as a single phosphatidylethanolamine (PE) lipid (Su et al, 2019). Structural comparison with the Mtb MmpL3 1-753 -LMNG complex identifies striking similarities in the binding poses of all four ligands (Figure 2C).…”

“…Segmentation of binding would permit accommodation of lipidic cargo in, and subsequent ll OPEN ACCESS Short Article manipulation into, diverse conformations. This flexibility is plausibly essential for translocation of substrates as large as TMM (molecular weight >1.4 kDa, Fujita et al, 2005), and likely underlies the lipid binding promiscuity observed for Msmg MmpL3 by native mass spectrometry (Su et al, 2019).…”

“…Atomic model building began by threading the Mtb MmpL3 1-753 sequence onto a previously reported crystal structure of the Msmg orthologue (PDB code: 6OR2, Su et al, 2019), using the Phyre2 web server (Kelley et al, 2015). The threaded structure was then docked into the 3.0 Å cryo-EM volume in UCSF Chimera (v.1.13.1, Pettersen et al, 2004), prior to further rigid body fitting in Coot (v.0.9.2-pre, Emsley et al, 2010).…”