New structure for TB lipid-uptake proteins offers drug-target clues

Tuberculosis (TB) remains a deadly infection caused by Mycobacterium tuberculosis (Mtb), killing more than a million people worldwide each year. A key to Mtb’s long-term survival in humans is its ability to scavenge fats from host cells; mammalian cell entry complexes (Mce1-4) help the bacterium import fatty acids and cholesterol and are especially important during the latent stage of infection. The proper functioning of these Mce complexes requires additional partners, including Mce-associated membrane (Mam) proteins and the lipid uptake coordinator LucA, so these accessory proteins have emerged as possible targets for new anti-TB drugs. The genes for four Mam proteins, Mam1A-1D, sit in the mce1 operon, while Mam3A-3B and Mam4A-4B are found in other operons; five orphaned mam (Omam) proteins have also been identified and appear relevant to the Mce system. Analysis of the sequences of Mam and Omam proteins suggests they share common secondary and tertiary structural elements even though their amino acid sequences differ. Under the leadership of corresponding author Rajaram Venkatesan, researchers set out to produce and study a Mam1A variant in the lab to learn how these proteins are built and how they associate with each other and with LucA.

To probe the shape and associations of Mam1A, the team made a recombinantly produced Mam1A variant and examined it using small-angle X-ray and neutron scattering. These scattering methods let researchers infer size, shape and assembly state of proteins in solution without needing a crystalline sample. The data indicate that Mam1A exists as a tetramer in solution and that two disulfide bridges are necessary for the stability of this tetrameric form. A disulfide bridge was also identified in Mam1C, suggesting that covalent bonds help stabilize these membrane-associated proteins. In addition to studying single proteins, the group used coexpression and copurification experiments to test interactions among the Mam proteins and LucA. Those experiments showed that Mam1A-1D and LucA can interact to form stable Mam1ABCD assemblies and larger Mam1ABCD-LucA complexes. Together these results map out basic structural features and direct interactions among Mam family members and LucA.

These structural and interaction findings matter because they provide concrete leads for understanding how Mce complexes are organized and stabilized. The discovery that Mam1A is a disulfide-stabilized tetramer and that Mam1C also contains a stabilizing disulfide bridge suggests specific structural elements that could be targeted by drugs or biochemical probes. Demonstrating stable Mam1ABCD and Mam1ABCD-LucA complexes clarifies which protein partners physically associate to support lipid uptake by Mce systems. Because Mce1-4 and their associated Mam and Omam proteins help Mtb import fatty acids and cholesterol during latent infection, blocking these interactions might undermine the bacterium’s ability to survive in host tissues. The authors say these results open the way to more detailed structural work to reveal precisely how Mam1ABCD and Mam1ABCD-LucA attach to Mce complexes and how that assembly can be disrupted, information that could be valuable in the search for new anti-TB strategies.