Variations of Subunit ε of the Mycobacterium tuberculosis F ₁ F _o ATP Synthase and a Novel Model for Mechanism of Action of the Tuberculosis Drug TMC207

Abstract

dThe subunit of bacterial F 1 F O ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of of Mycobacterium tuberculosis (Mt) F 1 F O ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mt 103-120 ). Mt is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic ␤ subunit. The C-terminal segment displays an ␣-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mt. A model for the interaction of TMC207 with both and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of and the c-ring, respectively. T19 and R37 of provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F 1 F O ATP synthase in M. tuberculosis.

Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

“…This correlates with results of recent computational studies of coevolution in protein complexes, which used interactions of ␥ and ⑀ as a test case (63,64). There are also indications that ⑀ inhibition also occurs in the enzyme of several species of Mycobacterium (65,66), and the CTD of most mycobacterial ⑀ subunits is ϳ17 residues shorter than that of E. coli, although different possible alignments make it uncertain how much of the hook and/or helix-2 are absent (67,68). Mycobacterial ATP synthase is the target of a new class of antibiotics, the diarylquinolines, and the lead drug, bedaquiline, has been approved for treatment of multidrug-resistant tuberculosis (25,69).…”

Energy Metabolism and Drug Efflux in Mycobacterium tuberculosis

“…Bedaquiline kills M. tuberculosis by interacting with the hydrophobic region of subunit c, as well as with subunit ε (48), of the F 1 F 0 -ATP synthase and does not cross-react with the human ATP synthase complex (56,58). Inhibition of c-ring rotation due to disruption in the c-ring:ε subunit interaction results in inhibition of ATP production and subsequent cell death (48). The efficacy of bedaquiline in clinical trials confirms that targeting energy metabolism during TB infection may be promising, particularly for nonreplicating organisms, as there is documented evidence that ATP is essential for the viability of nonreplicating persistent mycobacteria (20,55,59).…”

“…Rotation of subunits ε and ␥ is coupled to rotation of the c-ring upon proton translocation ( Fig. 1) (48,49). The mycobacterial F 1 F 0 -ATP synthase is encoded by a single operon, Rv1303-atpBEFHAGDC-Rv1312 (17).…”

“…The F 0 region is composed of 3 subunits designated a, b, and c, whereas the F 1 region is composed of 5 subunits designated ␣, ␤, ␦, ␥, and ε (45,46). ATP is synthesized by the ␣ 3 ␤ 3 -hexamer through rotation of the ␥-ε pair (48). Rotation of subunits ε and ␥ is coupled to rotation of the c-ring upon proton translocation ( Fig.…”

Asymmetric Synthesis and Absolute Configuration Assignment of a New Type of Bedaquiline Analogue

“…Because of its novel mechanism, bedaquiline does not have cross-resistance with current antituberculosis drugs. Many efforts have been made by scientists to locate the exact binding domain of bedaquiline in M. tuberculosis ATPase [5][6][7][8][9]. Most recently, in 2015, the X-ray crystal structures of a mycobacterial C 9 ring with and without a bedaquiline molecule were reported, and it was suggested that bedaquiline blocked the ion-binding sites of the c-subunits [10].…”