ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites

Abstract

The structures and functions of the components of ATP synthases, especially those subunits involved directly in the catalytic formation of ATP, are widely conserved in metazoans, fungi, eubacteria, and plant chloroplasts. On the basis of a map at 32.5-Å resolution determined in situ in the mitochondria of Trypanosoma brucei by electron cryotomography, it has been proposed that the ATP synthase in this species has a noncanonical structure and different catalytic sites in which the catalytically essential arginine finger is provided not by the α-subunit adjacent to the catalytic nucleotide-binding site as in all species investigated to date, but rather by a protein, p18, found only in the euglenozoa. A crystal structure at 3.2-Å resolution of the catalytic domain of the same enzyme demonstrates that this proposal is incorrect. In many respects, the structure is similar to the structures of F₁-ATPases determined previously. The α₃β₃-spherical portion of the catalytic domain in which the three catalytic sites are found, plus the central stalk, are highly conserved, and the arginine finger is provided conventionally by the α-subunits adjacent to each of the three catalytic sites found in the β-subunits. Thus, the enzyme has a conventional catalytic mechanism. The structure differs from previous described structures by the presence of a p18 subunit, identified only in the euglenozoa, associated with the external surface of each of the three α-subunits, thereby elaborating the F₁-domain. Subunit p18 is a pentatricopeptide repeat (PPR) protein with three PPRs and appears to have no function in the catalytic mechanism of the enzyme.

Keywords: ATP synthase; Trypanosoma brucei; catalytic domain; p18 subunit; structure.

Fig. 1.

Structure of the F₁-ATPase from T. brucei. The α-, β-, γ-, δ-, ε-, and p18-subunits are shown in red, yellow, blue, green, magenta, and cyan, respectively. (A and B) Side (A) and top (B) views in cartoon representation. (C–E) Side views in surface representation rotated 180° relative to A. (C) The bovine enzyme (12). (D and E) The T. brucei enzyme. In D, p18 has been omitted, and only additional regions not found in the bovine enzyme are colored; the rest of the structure is gray. The two additional sections in the α-subunit (red) interact with the p18-subunit. (E) p18 is present and is shown interacting with the α-subunit.

Fig. 2.

Conservation of the noncatalytic and catalytic nucleotide-binding sites in the F₁-ATPase from T. brucei. (A) The noncatalytic site in the α_DP-subunit superposed onto the equivalent site in the bovine enzyme (12). (B) The catalytic site in the β_DP-subunit superposed onto the equivalent site in the bovine enzyme. Residue αR386 is the catalytically essential arginine finger (equivalent to αR373 in the bovine protein). Residues contributed by α- and β-subunits are shown in red and yellow, respectively (with the bovine residues in muted colors), and the bound ADP molecules are in black in the T. brucei enzymes and in gray in the bovine enzymes. The green and red spheres represent magnesium ions and water molecules, respectively (in T. brucei only). The residue numbers in parentheses denote the equivalent bovine residues.

Fig. 3.

Structure of the p18-subunit of the F₁-ATPase from T. brucei, and its relation to a PPR protein. (A) A p18 subunit (cyan) in cartoon representation, folded into α-helices H1–H7, with an extended C-terminal region from residues 151–170, bound to the α_DP-subunit in solid representation (red). The N-terminal, nucleotide-binding, and C-terminal domains of the α-subunit are indicated by Crown, NBD, and C-ter, respectively; the bound ADP molecule is in black. (B) Comparison of the p18-subunit with an example PPR protein, the PPR10 protein from Z. mays (yellow) (57). PPR10 has 18 PPRs; the structures of PPRs 11–14 are shown (Fig. S3). The orange region represents the backbone of an eight-residue ribonucleotide bound to PPR10. The three PPRs in the p18-subunit correspond to H1 plus H2 (residues 20–28 and 33–45), H3 plus H4 (residues 52–64 and 78–93), and H5 plus H6 (residues 99–112 and 115–126). PPR10 has an additional α-helix, labeled 8, which together with α-helix 7 constitutes a fourth PPR.

Fig. 4.

Relationship of the crystallographic structure of the F₁-domain of the ATP synthase from T. brucei to an ECT map of the intact ATP synthase in situ in mitochondrial membranes from T. brucei. The subunits of the F₁-domain are colored as in Fig. 1. (A and B) Top (A) and side (B) views of the ECT map (gray), determined independently at 32.5-Å resolution with the crystallographic structure of the F₁-domain determined at 3.2-Å resolution docked manually inside the ECT map, with subunits α_DP and β_TP proximal to the peripheral stalk. (C and D) A published interpretation of the same ECT map proposing a structure of T. brucei F₁-ATPase in which the α-subunit is opened away from the central stalk, with the p18-subunit (not shown) contributing to the catalytic sites by providing the arginine finger residue (red circle) in D (42). The catalytic sites are indicated by green asterisks in C and by a green circle in D. PS, peripheral stalk of the enzyme. C and D modified from ref. .