New mutations in the mycobacterial ATP synthase: new insights into the binding of the diarylquinoline TMC207 to the ATP synthase C-ring structure

Abstract

TMC207 is a new antituberculous drug belonging to the diarylquinoline class which very efficiently inhibits the ATP synthase of mycobacteria such as Mycobacterium tuberculosis, one of the most important pathogens in the world. In order to map the amino acid residues involved in the binding of the drug, we have selected in vitro TMC207-resistant mutants from M. tuberculosis and diverse atypical mycobacteria. Six distinct mutations, Asp28 → Gly, Asp28 → Ala, Leu59 → Val, Glu61 → Asp, Ala63 → Pro, and Ile66 → Met, have been identified in the subunit c forming a C ring in the ATP synthase. They were studied by evaluating the levels of resistance that they confer in the selected clones and by using an isogenic complementation system in Mycobacterium smegmatis. The rates of increase of TMC207 MIC values (8- to 133-fold) were interpreted by constructing by homology modeling a structure of the mycobacterial C ring which was used for docking simulations with TMC207. Our results suggest that the residues found to be mutated in the resistant clones, together with a tyrosine specifically conserved at position 64 in mycobacteria, define a cleft located between two adjacent c subunits in the C ring. This cleft, which encompasses the proton-binding site (Glu61), is well fitted to bind TMC207 at the level of the bromoquinoline moiety, with the drug being anchored by several ionic, hydrogen, and halogen bonds with residues Glu61, Tyr64, and Asp28, respectively. These data shed light on the molecular interactions allowing TMC207 to bind specifically and efficiently at the level of the proton-binding site of the mycobacterial C ring.

Fig 1

Structure of TMC207 (R,S geometry) and subunit arrangements of [α₃β₃γε-ab₂-C ring] in the F-type ATP synthase. The region of interaction of the drug in the F-ATP synthase is shown by a black arrow pointing in subunit c to the location of the essential residue Asp or Glu allowing the ion transfer.

Fig 2

Multiple-sequence alignment of the subunits c from M. abscessus (Mabs), M. tuberculosis (Mtub), M. fortuitum (Mfor), M. smegmatis (Msme), E. coli (Ecol), Spirulina platensis (Spla), and I. tartaricus (Itar). Identical residues are marked with an asterisk. The gray shadow highlights variability between the amino acids from the mycobacterial species. The mutated positions previously reported in the drug-resistant mycobacterial strains (positions 28, 61, 63, and 66, M. tuberculosis numbering) are boxed. The mutated residues are indicated below the boxes. Amino acids shaded in black in the sequences of I. tartaricus and S. platensis correspond to the amino acids involved in the H⁺/Na⁺ binding in the subunits c of these species. Secondary-structure elements are indicated above the sequences.

Fig 3

Na⁺/H⁺ binding sites in the crystallographic structures of I. tartaricus (A) (35) and S. platensis (B) (34). The amino acids found in the ion coordination regions are shown in stick representation. Hydrogen bonds are represented by dashed lines. (C) Structural model of the same region in M. tuberculosis (this study). The amino acids shown in stick representation (apart from Tyr64) are those implicated in TMC207 resistance.

Fig 4

Optimal docking result obtained for the R,S stereoisomer of TMC207 in the C ring of M. tuberculosis. The polypeptide chains forming the binding cleft are shown in ribbon representation. The amino acid residues contributing to the binding region are shown in stick representation. Oxygen and nitrogen atoms are indicated in red and dark blue, respectively. The TMC207 molecule is represented by using CPK colors, and the bromine atom is shown as a sphere. Hydrogen and halogen bonds are represented by dotted lines.