A systematic assessment of mycobacterial F₁‐ATPase subunit ε’s role in latent ATPase hydrolysis

Abstract

In contrast to most bacteria, the mycobacterial F1FO‐ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) does not perform ATP hydrolysis‐driven proton translocation. Although subunits α, γ and ε of the catalytic F1‐ATPase component α3:β3:γ:ε have all been implicated in the suppression of the enzyme’s ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ε into focus by generating the recombinant Mycobacterium smegmatis F1‐ATPase (MsF1‐ATPase), whose 3D low‐resolution structure is presented, and its ε‐free form MsF1αβγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ε’s suppression of ATPase activity. Deletion of four amino acids at ε’s N terminus, mutant MsF1αβγεΔ2‐5, revealed similar ATP hydrolysis as MsF1αβγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N‐terminal ε‐mutants, the importance of the first N‐terminal residues of mycobacterial ε in structure stability and latency is described. Engineering ε’s C‐terminal mutant MsF1αβγεΔ121 and MsF1αβγεΔ103‐121 with deletion of the C‐terminal residue D121 and the two C‐terminal ɑ‐helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α3β3‐headpiece and its function in ATP hydrolysis inhibition. Finally, we applied the tools developed during the study for an in silico screen to identify a novel subunit ε‐targeting F‐ATP synthase inhibitor.

The Unique C-Terminal Extension of Mycobacterial F-ATP Synthase Subunit α Is the Major Contributor to Its Latent ATP Hydrolysis Activity

“…The purification protocol and 12% SDS-PAGE gel were replicated at least three times, and results represented in the elution diagram and gel remained consistent. (B) Densitometric analysis of the ␥ to ratio of MsF 1 -␣ Δ514-549 ␤␥ revealed a 1:0.3 ratio, identical to that of the WT enzyme (16) and demonstrating the correct stoichiometric subunit ratio. (C) Recombinant mutants were tested for their ATP hydrolysis rate.…”

“…First, the ␣ CTD -deleted M. smegmatis F 1 -ATPase mutant, MsF 1 -␣ Δ514-549 ␤␥, was engineered using the recently generated template of the atp genes AGDC, encoding subunits ␣:␤:␥: within the pYUB1049 vector (16,18) and the primers listed in Table S1 FIG 1 Amino acid sequence alignment of subunit ␣ of different mycobacterial organisms in comparison with Homo sapiens, Escherichia coli, and G. stearothermophilus. The sequence alignment of subunit ␣ of the following organisms: H. sapiens (UniProt ID P25705-2), E. coli (UniProt ID P0ABB0), G. stearothermophilus (UniProt ID P42005), M. tuberculosis (UniProt ID P9WPU7), M. smegmatis (UniProt ID A0R202), and Mycobacterium bovis (UniProt ID A1KI96) were obtained from the UniProt database (30) and imported into Jalview (31).…”

“…in the supplemental material. The linearized pYUB1049 vector was amplified (19), and the two DNA fragments were incorporated as previously published (16). To ease purification, a His 6 tag was added to the N terminus of the ␤ subunit (18).…”

“…To ease purification, a His 6 tag was added to the N terminus of the ␤ subunit (18). Protein purification was performed as mentioned previously published (16), with an MsF 1 -␣ Δ514-549 ␤␥ in proper stoichiometry and an ␣ Δ514-549 band running faster than its wild-type (WT) counterpart, revealing the successful deletion (Fig. 2A and B).…”

“…2A and B). Subsequently, continuous ATP hydrolysis assay was performed according to previously published methods (16,20,21). ATPase activity of 0.05 Ϯ 0.001 mol min Ϫ1 (mg of protein) Ϫ1 was calculated for WT MsF 1 -ATPase (Fig.…”