Abstract
Oxidative phosphorylation is an essential pathway in mycobacteria and inhibitors of this pathway are actively being developed and improved. The type II NADH dehydrogenase (NDH-2) is a promising target for development of anti-mycobacterial compounds due to its importance in mycobacterial oxidative phosphorylation and its absence of mammalian homologues. However, lack of structural insight into binding of inhibitors to the enzyme has prevented efficient optimization of lead compounds. We present the structure of NDH-2 from Mycobacterium smegmatis determined by electron cryomicroscopy (cryo-EM), both alone and bound to 2-mercaptoquinazolinone, a validated inhibitor. The structure reveals that the active form of mycobacterial NDH-2 is dimeric with the dimerization interface stabilized by an extended C-terminal alpha helix. The dimerization interface of mycobacterial NDH-2 appears to be significantly larger than those of other published prokaryotic NDH-2 dimer structures, instead resembling eukaryotic NDH-2 dimers. As a peripheral membrane protein purified after detergent solubilization, a prominent detergent micelle is observed on the membrane-binding surface of the enzyme, which was critical for determining the high-resolution cryoEM map of the sub-100 kDa protein assembly. We found that 2-mercaptoquinazolinone inhibits mycobacterial NDH-2 in the native membrane at sub-micromolar concentrations and binds to the quinone binding pocket, which is a highly conserved site among mycobacterial species. Our results provide insight into the structural organization of mycobacterial NDH-2 and how it can be inhibited to kill mycobacteria.The published version is not available in full-text in OUR Archive. Where available, a link to the published version is provided (check the DOI and/or the Files and links section). The full-text item may be open access on the publisher's website. An earlier version of the work (such as authors' accepted manuscript following peer-review or unreviewed preprint/author's original version) may be available in the Files and links section of this record. Alternatively, readers may have subscription access to the full-text from the publisher.