Bioenergetics
The uniqueness of subunit α of mycobacterial F-ATP synthases: An evolutionary variant for niche adaptation

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The F1F0 -ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear. Here, we report that a Mycobacterium-specific, 36-amino acid long C-terminal domain in the nucleotide-binding subunit α (Mtα) of F-ATP synthase suppresses its ATPase activity and determined the mechanism of suppression. First, we employed vesicles to show that in intact membrane-embedded mycobacterial F-ATP synthases deletion of the C-terminal domain enabled ATPase and proton-pumping activity. We then generated a heterologous F-ATP synthase model system, which demonstrated that transfer of the mycobacterial C-terminal domain to a standard F-ATP synthase α subunit suppresses ATPase activity. Single-molecule rotation assays indicated that the introduction of this Mycobacterium-specific domain decreased the angular velocity of the power-stroke after ATP binding. Solution X-ray scattering data and NMR results revealed the solution shape of Mtα and the 3D structure of the subunit α C-terminal peptide 521PDEHVEALDEDKLAKEAVKV540 of M. tubercolosis (Mtα(521–540)), respectively. Together with cross-linking studies, the solution structural data lead to a model, in which Mtα(521–540) comes in close proximity with subunit γ residues 104–109, whose interaction may influence the rotation of the camshaft-like subunit γ. Finally, we propose that the unique segment Mtα(514–549), which is accessible at the C terminus of mycobacterial subunit α, is a promising drug epitope.

ATP synthase
bioenergetics
F1FO-ATPase
membrane protein
tuberculosis
F-ATP synthase
Mycobacterium
subunit α

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This work was supported in part by the Ministry of Education, Singapore Grants MOE2011-T2-2-156 and ARC 18/12, Ministry of Health, Singapore, NMRC Grant CBRG12nov049 (to G. G.), Ministry of Health, Singapore Grant MOE2012-T2-1-101 (to T. W.), and National Institute of Health Grant R01GM097510 (to W. F.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains supplemental Video S1 and Table S1.

The atomic coordinates and structure factors (code 5WY7) have been deposited in the Protein Data Bank (http://wwpdb.org/).

1

These authors contributed equally to the results of this work.

2

Supported by a research scholarship from Nanyang Technological University.

3

Supported by a research scholarship from the Ministry of Education.