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• First purification protocol of an intact mycobacterial F-ATP synthase. • First low resolution structure of an entire mycobacterial F-ATP synthase. • Purification of the fused mycobacterial b -δ 24-446 construct. • The novel 111-residues domain of mycobacterial subunit δ. • The potential of mycobacterial subunit δ as a drug target. In contrast to other prokaryotes, the Mycobacterial F 1 F O ATP synthase (α 3 :β 3 :γ:δ:ε: a : b : b' : c 9) 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 α 3 :β 3 :γ:ε-domain and c subunit ring were resolved. Here, we report the first purification protocol of the intact M. smegmatis F 1 F O ATP synthase including the F 1 -domain, the entire membrane-embedded F O 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 F 1 F O ATP synthase by electron microscopy (EM) and single particle analysis. Expression and purification of the fused mycobacterial b -δ 24-446 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 α 3 :β 3 :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 α 3 β 3 -headpiece and its potential as a drug target are discussed. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • 3D reconstruction from electron micrographs of the α chi 3 :β 3 :γ complex. • Low resolution solution structure of the mycobacterial F-ATP synthase subunit α. • Critical residues of subunit ε for energy coupling of mycobacterial F-ATP synthase. • Residue 121 of mycobacterial subunit ε affects MgADP inhibition. • Differences of the N-terminus of mycobacterial ε to its prokaryotic counterparts. Abstract The Mycobacterium tuberculosis (Mtb) F 1 F O -ATP synthase (α 3 :β 3 :γ:δ:ε: a : b : b' : c 9) is an essential enzyme that supplies energy for both the aerobic growing and the hypoxic dormant stage of the mycobacterial life cycle. Employing the heterologous F-ATP synthase model system α chi 3 :β 3 :γ we showed previously, that transfer of the C-terminal domain (CTD) of Mtb subunit α (Mt α 514-549) to a standard F-ATP synthase α subunit suppresses ATPase activity. Here we determined the 3D reconstruction from electron micrographs of the α chi 3 :β 3 :γ complex reconstituted with the Mtb subunit ε (Mt ε), which has been shown to crosstalk with the CTD of Mt α. Together with the first solution shape of Mtb subunit α (Mt α), derived from solution X-ray scattering, the structural data visualize the extended C-terminal stretch of the mycobacterial subunit α. In addition, Mt ε mutants Mt εR62L, Mt εE87A, Mt ε 6-121 , and Mt ε 1-120 , reconstituted with α chi 3 :β 3 :γ provided insight into their role in coupling and in trapping inhibiting MgADP. NMR solution studies of Mt εE87A gave insights into how this residue contributes to stability and crosstalk between the N-terminal domain (NTD) and the CTD of Mt ε. Analyses of the N-terminal mutant Mt ε 6-121 highlight the differences of the NTD of mycobacterial subunit ε to the well described Geobacillus stearothermophilus or Escherichia coli counterparts. These data are discussed in context of a crosstalk between the very N-terminal amino acids of Mt ε and the loop region of one c subunit of the c -ring turbine for coupling of proton-translocation and ATP synthesis activity. [ABSTRACT FROM AUTHOR]