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 WzmWzt_Mtb to uncover critical steps of Mtb LLG translocation. Notably, densities suggestive of the mycobacterial-derived LLG substrates were observed in the structure of WzmWzt_Mtb, 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 WztWzm_Mtb 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 (IF_N) 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.