Insight into the structure and interactions of the M. tuberculosis Mce-associated membrane proteins Mam1A-1D

Abstract

Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb), leading to more than a million human deaths every year. Mammalian cell entry complexes (Mce1-4) play an essential role in the survival of Mtb during the latent stage by mediating the import of lipids, including fatty acids and cholesterol, from the host. The proper functioning of Mce-complexes requires additional proteins such as Mce-associated membrane (Mam) proteins and lipid uptake coordinator (LucA), thus making them potential candidates for the development of anti-TB drugs. Four Mam (Mam1A-1D) proteins are coded from the mce1 operon and two from the mce3 (Mam3A-3B) and mce4 (Mam4A-4B) operons. In addition, five orphaned mam (Omam) proteins have been identified, which are not part of the mce operons but are functionally relevant for the Mce complexes. Analysis of the sequences of Mam/Omam proteins suggests that they share many common secondary and tertiary structural elements despite the low sequence identity between them. Here, we have characterized a recombinantly produced Mam1A variant by small-angle X-ray and neutron scattering. The studies indicate that Mam1A is tetrameric in solution with two disulfide bridges necessary for the stability of Mam1A. Similarly, a disulfide bridge has also been identified in Mam1C. Furthermore, through coexpression and copurification, we demonstrate that Mam1A-1D and LucA interact to form stable Mam1ABCD as well as Mam1ABCD-LucA complexes. The results obtained pave the way for further understanding how the Mam1ABCD and Mam1ABCD-LucA complexes are organized and interact with the Mce complexes, leading to their stabilization.