Atomic solution structure of Mycobacterium abscessus F-ATP synthase subunit epsilon and identification of Ep1MabF1 as a targeted inhibitor

Mycobacterium abscessus (Mab) is a nontuberculous mycobacterium of increasing clinical relevance. The rapidly growing opportunistic pathogen is intrinsically multi-drug-resistant and causes difficult-to-cure lung disease. Adenosine triphosphate, generated by the essential F1Fo ATP synthase, is the major energy currency of the pathogen, bringing this enzyme complex into focus for the discovery of novel antimycobacterial compounds. Coupling of proton translocation through the membrane-embedded F-o sector and ATP formation in the F-1 headpiece of the bipartite F1Fo ATP synthase occurs via the central stalk subunits gamma and epsilon. Here, we used solution NMR spectroscopy to resolve the first atomic structure of the Mab subunit epsilon (Mab epsilon), showing that it consists of an N-terminal beta-barrel domain (NTD) and a helix-loop-helix motif in its C-terminal domain (CTD). NMR relaxation measurements of Mab epsilon shed light on dynamic epitopes and amino acids relevant for coupling processes within the protein. We describe structural differences between other mycobacterial epsilon subunits and Mab epsilon's lack of ATP binding. Based on the structural insights, we conducted an in silico inhibitor screen. One hit, Ep1MabF1, was shown to inhibit the growth of Mab and bacterial ATP synthesis. NMR titration experiments and docking studies described the binding epitopes of Ep1MabF1 on Mab epsilon. Together, our data demonstrate the potential to develop inhibitors targeting the 8 subunit of Mab F1Fo ATP synthase to interrupt the coupling process.