Neelagandan Kamariah, Roland G. Huber, Wilson Nartey, Shashi Bhushan, Peter J. Bond, Gerhard Grüber, Structure and subunit arrangement of Mycobacterial F1FO ATP synthase and novel features of the unique mycobacterial subunit δ, Journal of Structural Biology, Volume 207, Issue 2, 2019, Pages 199-208, ISSN 1047-8477, https://doi.org/10.1016/j.jsb.2019.05.008.
In contrast to other prokaryotes, the Mycobacterial F1FO ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) is essential for growth. The mycobacterial enzyme is also unique as a result of its 111 amino acids extended δ subunit, whose gene is fused to the peripheral stalk subunit b. Recently, the crystallographic structures of the mycobacterial α3:β3:γ:ε-domain and c subunit ring were resolved. Here, we report the first purification protocol of the intact M. smegmatis F1FO ATP synthase including the F1-domain, the entire membrane-embedded FO sector, and the stator subunits b’ and the fused b-δ. This enzyme purification enabled the determination of the first projected 2D- and 3D structure of the intact M. smegmatis F1FO ATP synthase by electron microscopy (EM) and single particle analysis. Expression and purification of the fused mycobacterial b-δ24-446 construct, excluding the membrane-embedded N-terminal amino acids, provided insight into its secondary structure. By combining these data with homology and ab-initio modeling techniques, a model of the mycobacterial peripheral stalk subunits b-δ and b’ was generated. Superposition of the 3D M. smegmatis F-ATP synthase EM-structure, the α3:β3:γ:ε and c-ring, and the derived structural models of the peripheral stalk enabled a clear assignment of all F-ATP synthase subunits, in particular with respect to the unique mycobacterial peripheral stalk subunit b’ and the elongated δ fused with subunit b. The arrangement of δ relative to the N-termini of the catalytic α3β3-headpiece and its potential as a drug target are discussed.
We are grateful to Dr. S. S. M. Malathy (School of Biological Sciences, NTU) for the art work of Figs. 1 and 8. W. Nartey is grateful to receive a PhD scholarship from the Singapore International Graduate Award (SINGA). This research was supported by the National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF–CRP18–2017–01; Lead-PI G.G.). The computational work for this article was partially performed on resources of the National Supercomputing Centre, Singapore (https://www.nscc.sg).