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

Abstract

The FF -ATP (F-ATP) synthase is essential for growth of , 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-specific, 36-amino acid long C-terminal domain in the nucleotide-binding subunit α (α) 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 -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 ofα and the 3D structure of the subunit α C-terminal peptide PDEHVEALDEDKLAKEAVKV of (α(521-540)), respectively. Together with cross-linking studies, the solution structural data lead to a model, in which α(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 segmentα(514-549), which is accessible at the C terminus of mycobacterial subunit α, is a promising drug epitope.

The NMR solution structure of Mycobacterium tuberculosis F‐ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine

“…7D). This is in line with recent observations of deletion mutants of the C-terminus (Da 514-549 ) and the novel loop region (Dc166-179) of the mycobacterial ɑ and c subunit, respectively (Dc166-179), both resulting in significant reduction in ATP synthesis but without major differences in cell growth compared to WT [23,26]. As pointed out above, in rich media glucose provides the bacterium with sufficient carbohydrates, increasing the metabolic flux through the Krebs cycle, and generating higher yields of NADH and ATP.…”

“…(C) A structural model of the α chi 3 β 3 γ‐complex generated based on the G. stearothermophilus F 1 ‐ ATP ase structure ( PDB ID : 4XD7 ). The solution shape of α chi (green sphere) was superimposed on to the Gs α structure (16) (green) and the NMR solution structure of the C‐terminal peptide Mt α 521‐540 (green) was placed in the extra density of the SAXS ‐shape. The Mt ε (magenta) inside the α chi 3 β 3 γ‐complex was modeled to have extended Mt ε CTD .…”

“…The plasmid containing the α chi βγ genes was used as a template for site‐directed mutagenesis and to generate the mutant (α chi ‐S400C) 3 β 3 γ. The entire plasmid (pET‐24b vector) was amplified by KAPA HiFi DNA polymerase (KAPA Biosystems) using forward 5′–CGCCCAATTCGGC TGC GACCTCGATAAA–3′ and the reverse primer 5′–TTTATCGAGGTC GCA GCCGAATTGGGCG–3′.…”

“…The plasmid containing the a chi bc genes [26] was used as a template for site-directed mutagenesis and to generate the mutant (a chi -S400C) 3 b 3 c. The entire plasmid (pET-24b vector) was amplified by KAPA HiFi DNA polymerase (KAPA Biosystems) using forward 5 0 -CGCCCAATT CGGCTGCGACCTCGATAAA-3 0 and the reverse primer 5 0 -TTTATCGAGGTCGCAGCCGAATTGGGCG-3 0 . The amplified PCR product was purified and DpnI digestion was performed to digest the methylated parental plasmid.…”

The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs

“…However, the sequence register could not be determined unambiguously, and so this segment is modeled as unknown and numbered 1512-1522. In a peptide representing residues 521-540, it has been shown by solution nuclear magnetic resonance (NMR) that residues 526-539 are α-helical (but no coordinates are available) and on the basis of structure prediction that this α-helical structure prevailed in the intact protein (19). However, the current prediction of intrinsic disorder in the entire C-terminal region from residues 512-548 (SI Appendix, Fig.…”