Tuberculosis is the seventh most common cause of death globally. The complete genome sequence of Mycobacterium tuberculosis, its causative agent, has allowed identification of several stress response genes that contribute to pathogenicity. Among these, the membrane protein Rv0899 is a virulence factor that confers adaptation of M. tuberculosis to the acidic environment of the phagosome. Its gene is restricted to pathogenic mycobacteria associated with TB and other TB-related diseases and, thus, is an attractive candidate for the development of anti-TB chemotherapeutic agents. We have determined the three dimensional structure and dynamics of residues 73-326 of this 326-residue protein. In contrast to the original predictions, residues 73-326 form a globular structure, which encompasses two independently folded domains, with mixed α/β-secondary structure, connected by a flexible linker. The central B domain (residues 80-195) reveals for the first time the fold of a BON homology domain associated with bacterial osmotic shock resistance, nodulation-specificity and lipid-binding proteins. The C-terminal domain (residues 205-326) adopts the typical fold of peptidoglycan-binding domains, and also binds peptidoglycan suggesting a periplasmic localization for this part of the protein. Residues 1-73 contain a 20-residue hydrophobic sequence that forms a transmembrane anchor. The overall architecture of the protein, its ligands, and the unexpected structure of the B domain make it difficult to reconcile a porin activity with its central domain but suggest alternative modes of membrane association.[This research was supported by the National Institutes of Health (AI074805, GM075917). It utilized the NMR Facilities at the Sanford Burnham Medical Research Institute and at the University of California, San Diego, supported by grants from the National Institutes of Health (CA030199, EB002031, RR23773)].