Oligomerization is required for channel formation of MctB and for copper resistance of Mycobacterium tuberculosis

TL;DR

This study reveals that MctB, a protein in tuberculosis bacteria, forms oligomers to create channels that help the bacteria resist copper toxicity.

Contribution

The study identifies the structural and functional role of MctB oligomerization in copper resistance of Mycobacterium tuberculosis.

Findings

MctB forms oligomers that are essential for its membrane insertion and pore formation.

Oligomeric MctB can translocate ions across membranes, contributing to copper resistance.

The N-terminal helix of MctB is crucial for its export, membrane association, and function.

Abstract

The mammalian immune system kills bacterial pathogens including Mycobacterium tuberculosis by increasing copper uptake into the phagosome of infected macrophages. Rv1698 was previously identified as a membrane-spanning channel protein. The rv1698 deletion mutant of M. tuberculosis accumulated 100-fold more copper and lungs of infected guinea pigs had a 1000-fold reduced bacterial burden compared to the WT strain. Thus, Rv1698 is an important virulence factor and was named mycobacterial copper transport protein B (MctB). However, the mechanism by which MctB confers copper resistance is unknown. Here, we solved the crystal structure of MctB which revealed a ∼7 nm long helix followed by a large globular Rossmann-like domain. Subsequent experiments showed that the N-terminal hydrophobic helix is essential for MctB export into the periplasm, for its membrane association and for its function in copper resistance. Analytical size-exclusion chromatography and gel electrophoresis showed that monomeric water-soluble MctB is in equilibrium with oligomers of molecular masses of up to ∼400 kDa. Self-assembly of MctB is induced by detergents. Importantly, only oligomeric MctB inserts into membranes in lipid bilayer experiments and forms open membrane-spanning pores capable of translocating ions. Oligomeric MctB complexes were visualized by electron microscopy. Deciphering the atomic structure of oligomeric MctB will be instrumental in understanding the molecular mechanism by which MctB contributes to copper resistance in mycobacteria.

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