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

Abstract

Mycobacterial F₁F_o-ATP synthases (α₃:β₃:γ:δ:ε:a:b:b':c₉ ) are incapable of ATP-driven proton translocation due to their latent ATPase activity. This prevents wasting of ATP and altering of the proton motive force, whose dissipation is lethal to mycobacteria. We demonstrate that the mycobacterial C-terminal extension of nucleotide-binding subunit α contributes mainly to the suppression of ATPase activity in the recombinant mycobacterial F₁-ATPase. Using C-terminal deletion mutants, the regions responsible for the enzyme's latency were mapped, providing a new compound epitope.

Keywords: ATP hydrolysis; F-ATP synthase; Mycobacterium; bioenergetics; subunit α; tuberculosis.

FIG 1

Amino acid sequence alignment of subunit α of different mycobacterial organisms in comparison with Homo sapiens, Escherichia coli, and G. stearothermophilus. The sequence alignment of subunit α 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). Alignment of the sequences was performed using ClustalWS (32). Thereafter, the calculation of the percentage of identity was performed and presented in darker to lighter shades of blue, representing the most homologous to the least homologous. As highlighted in red, the C-terminal extension was observed specifically in mycobacteria and not in other species. As previously studied, the α-helix is present from V525 to V538 (according to M. tuberculosis amino acid numbering). For reference, the α-helix present in the C terminus is presented by a green cylinder, and the region showing no secondary structure is denoted by a single black line.

FIG 2

Characterization of the recombinant MsF₁-α_ΔCTD mutants. (A) Fractions from ion exchange were pooled and subjected to size-exclusion chromatography. The recombinant proteins showed consistency in elution at ∼11.6 ml, and their integrity and constituents were confirmed on a 12% SDS-PAGE gel (inset). The subunits are labeled, where α* refers to subunit α and its mutants β, γ, and ε, which correspond to ∼60, 54 , 35, and 10 kDa, respectively. The corresponding proteins are as labeled: lane 1, MsF₁-ATPase; lane 2, MsF₁-α_Δ514-549βγε; lane 3, MsF₁-α_Δ523-549βγε; and lane 4, MsF₁-α_Δ538-549βγε. 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 γ to ε ratio of MsF₁-α_Δ514-549βγε 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. The decrease in NADH absorption at 340 nm is plotted against the progressing time. MsF₁-α_Δ514-549βγε showed a significant increase in ATP hydrolysis (red triangle). On the other hand, MsF₁-α_Δ523-549βγε (purple diamond) and MsF₁-α_Δ538-549βγε (green asterisk) showed lesser ATP hydrolysis than MsF₁-α_Δ514-549βγε. To calculate the specific activity, the initial rate was used (solid lines), and their calculated specific activities and standard error of regression slope (S_b1) were 3.31 ± 0.18, 1.54 ± 0.03, and 1.33 ± 0.01 μmol min⁻¹ (mg of protein)⁻¹.

FIG 3

A proposed mechanism of ATP hydrolysis inhibition. (A) Part of the T. brucei F₁-ATPase crystal structure (PDB ID 6F5D ) (22) and a further zoom to highlight the proximity of its extended subunit α C terminus and ADP. The T. brucei C-terminal residues 536 to 539 (red) form an α-helical turn, followed by a random region (540 to 544) and an α-helix (546 to 558) that come close to the ADP. A conformational alteration could bring R558 closer to ADP to generate a hydrogen bond with ADP, or one of the C-terminal residues, not resolved in the structure, could come in proximity to the nucleotide. We predict that the C-terminal residues 538 to 549 of mycobacterial subunit α may come in close proximity to the ADP to stabilize the inhibited state. Subunits α, β, and γ and the T. brucei-specific p18 are shown in green, orange, yellow, and cyan, respectively. The figure was generated via PyMOL (33).