Structure of an endogenous mycobacterial MCE lipid transporter

Abstract

To replicate inside human macrophages and cause the disease tuberculosis, Mycobacterium tuberculosis (Mtb) must scavenge a variety of nutrients from the host1,2. The Mammalian Cell Entry (MCE) proteins are important virulence factors in Mtb1,3, where they are encoded in large gene clusters and have been implicated in the transport of fatty acids4–7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and how the ~10 proteins encoded in a mycobacterial mce gene cluster might assemble to transport cargo across the cell envelope remains unknown. Here we report the cryo-EM structure of the endogenous Mce1 fatty acid import machine from Mycobacterium smegmatis, a non-pathogenic relative of Mtb. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex, long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Unexpectedly, our structural data revealed the presence of a previously unknown subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated fatty acid import across the mycobacterial cell envelope.

Protein target highlights in CASP15: Analysis of models by structure providers

“…At the level of quaternary structure, the top five predicted models aligned well with the experimental structure with average α‐carbon RMSDs of 0.75 Å for the portal, 0.88 Å for the ring, 1.64 Å for the ABC transporter, and 3.34 Å for the needle. For the portal domain, the top five predictions were similar to the cryo‐EM structure, with additional predicted segments for parts of the Mce1C, Mce1D, and Mce1F C‐termini that were unresolved in the cryo‐EM map 46 (Figure 1C). The ring domain was also well predicted in all five models with minor deviations in the loops lining the central pore (Figure 4D).…”

“…Our structure revealed how proteins from the mce1 operon assemble to form an unusual ATP‐binding‐cassette (ABC) transporter complex with a long hydrophobic tunnel for protected lipid transport across the bacterial cell envelope (Figure 4A). 46 The Mce1 complex consists of 10 protein subunits: YrbE1A, YrbE1B, Mce1A, Mce1B, Mce1C, Mce1D, Mce1E, Mce1F, and two copies of MceG. Mce1 contains four major parts: (1) the portal, a globular domain at the top of the needle; (2) the needle, a curved hydrophobic tunnel created by a superhelix of 6 α‐helical segments; (3) the ring, formed by a heterohexamer of MCE domains; and (4) the ABC transporter, which consists of YrbE1AB permease and MceG ATPase subunits (Figure 4A).…”

“…Our structure revealed how proteins from the mce1 operon assemble to form an unusual ATP-binding-cassette (ABC) transporter complex with a long hydrophobic tunnel for protected lipid transport across the bacterial cell envelope (Figure 4A). 46 4A).…”

Rapid proteome-wide prediction of lipid-interacting proteins through ligand-guided structural genomics

“…A second key limitation is that the model only identifies LBPs that are embedded within monomeric proteins. If a lipid binds to the protein's surface (such as with ApoA1 and ApoE), or if the binding site is formed upon the oligomerization of one or more subunits (an example is the recently reported MCE transport system 54 ), the classifier is unable to detect these as LBPs. Regardless, the high precision suggests that SLiPP is well suited as a tool to facilitate the identification of novel lipid interacting proteins and complements existing low-throughput discovery methods.…”

Flipping the switch: dynamic modulation of membrane transporter activity in bacteria

“…However, upon binding WatS1, WatABO specifically increases its ability to efflux acetate, but not other tested organic acids [92]. In addition, a recent structure of a novel ABC transporter for lipid uptake from Mycobacterium tuberculosis revealed a previously unknown but conserved small transmembrane protein, LucB, that was proposed to be involved in regulating activity [93]. Overall, these data suggest that small proteins can play important roles in modulating the activities of various types of transporter.…”