Recognition and mechanism of M. tuberculosis cell wall galactan transport.

Mycobacterial-derived lipid-linked galactan (LLG) is an essential component of the Mycobacterial tuberculosis (Mtb) cell wall core arabinogalactan (AG), a well-established virulence factor and target of anti-tubercular drugs. Cross-membrane translocation of LLG is energetically unfavorable and is therefore catalyzed by WzmWzt, a prominent member of the polysaccharide ABC transporter sub-family. Here we present cryo-EM structures of WzmWztMtb to uncover critical steps of Mtb LLG translocation. Notably, densities suggestive of the mycobacterial-derived LLG substrates were observed in the structure of WzmWztMtb, indicating that the substrate may bind through two Wzm and two Wzt subunits. This proposed LLG binding is fulfilled by a fully upward extension of the Wzt gate helix, which serves as a critical step to prime Wzt for dimerization. The ATP-bound structure revealed a remarkable downward movement of the gate helix, together with the Wzt dimerization. The third, DDM/ATP-bound structure captured WztWzmMtb in an intermediate state with two DDM molecules in the Wzm central cavity. Using two conditional mycobacterial mutants for functional analyses, we have identified residues on the Wzm N-terminal interface helix (IFN) and on the Wzt helical domain are essential for LLG recognition and translocation in vivo. These data provide a clear structural basis for the development of Mtb cell wall targeted therapeutics for global tuberculosis treatment.