ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites Martin G. Montgomerya,1, Ondrej Gahuraa,b,1, Andrew G. W. Lesliec, Alena Zíkováb, and John E. Walkera,2 aThe Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge CB2 0XY, United Kingdom; bInstitute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005 Ceské Budejovice, Czech Republic; and cThe Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom Contributed by John E. Walker, December 18, 2017 (sent for review December 1, 2017; reviewed by Thomas M. Duncan and Wayne D. Frasch) The structures and functions of the components of ATP synthases, and penetrates into the α3β3-domain, where the catalytic sites of especially those subunits involved directly in the catalytic forma- the enzyme are found at three of the interfaces of α- and tion of ATP, are widely conserved in metazoans, fungi, eubacteria, β-subunits. The penetrant region of the central stalk is an and plant chloroplasts. On the basis of a map at 32.5-Å resolution asymmetric α-helical coiled coil, and its rotation inside the α3β3- determined in situ in the mitochondria of Trypanosoma brucei by domain takes each catalytic site through a series of conforma- electron cryotomography, it has been proposed that the ATP syn- tional changes that lead to the binding of substrates and the thase in this species has a noncanonical structure and different formation and release of ATP. catalytic sites in which the catalytically essential arginine finger During ATP hydrolysis in the experimentally detached F1- is provided not by the α-subunit adjacent to the catalytic domain, the direction of rotation, now driven by energy released nucleotide-binding site as in all species investigated to date, but from the hydrolysis of ATP, is opposite to the synthetic sense. rather by a protein, p18, found only in the euglenozoa. A crystal Extensive structural analyses, mostly by X-ray crystallography at structure at 3.2-Å resolution of the catalytic domain of the same atomic resolution, have shown that the F1-domains of the en- enzyme demonstrates that this proposal is incorrect. In many re- zymes from bovine (3–23) and yeast (24–30) mitochondria, spects, the structure is similar to the structures of F1-ATPases de- chloroplasts (31, 32), and eubacteria (33–39) are highly con- α β termined previously. The 3 3-spherical portion of the catalytic served. Not only is there conservation of the subunit composi- domain in which the three catalytic sites are found, plus the cen- tions of the α3β3-domain and the central stalk (γ1e1 in eubacteria tral stalk, are highly conserved, and the arginine finger is provided γ δ α and chloroplasts, and 1 1 plus an additional unique subunit, conventionally by the -subunits adjacent to each of the three confusingly called e, attached to the δ-subunit in mitochondria catalytic sites found in the β-subunits. Thus, the enzyme has a orthologs), but also the sequences of subunits are either highly conventional catalytic mechanism. The structure differs from pre- conserved or absolutely conserved in many key residues. This vious described structures by the presence of a p18 subunit, iden- extensive conservation includes residues in catalytic interfaces tified only in the euglenozoa, associated with the external surface and in the catalytic sites themselves. In the β-subunits, they 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 Significance mechanism of the enzyme. Mitochondria generate the cellular fuel ATP to sustain complex ATP synthase | Trypanosoma brucei | p18 subunit | catalytic domain | structure life. Production of ATP depends on the oxidation of energy-rich compounds to produce the proton motive force (pmf), a chemical potential difference for protons, across the inner he ATP synthases, also known as F-ATPases or F1Fo- TATPases, are multisubunit enzyme complexes found in membrane. The pmf drives the ATP synthase to synthesize ATP energy-transducing membranes in eubacteria, chloroplasts, and via a mechanical rotary mechanism. The structures and func- mitochondria (1, 2). They make ATP from ADP and phosphate tions of the protein components of this molecular machine, under aerobic conditions using a proton-motive force (pmf), especially those involved directly in the catalytic formation of generated by respiration or photosynthesis, as a source of energy. ATP, are widely conserved in metazoans, fungi, and eubacteria. Here we show that the proposal that this conservation does To date, studies of the subunit compositions, structures, and Trypanosoma brucei mechanism of the ATP synthases have been confined mainly to not extend to the ATP synthase from ,a member of the euglenozoa and the causative agent of sleeping the vertebrates, especially humans and bovines, and to various sickness in humans, is incorrect. fungi, eubacteria, and chloroplasts of green plants. These studies have established the conservation of the central features of these Author contributions: A.Z. and J.E.W. designed research; M.G.M. and O.G. performed rotary machines. They are all membrane-bound assemblies of research; M.G.M., A.G.W.L., and J.E.W. analyzed data; J.E.W. wrote the paper; and multiple subunits organized into membrane-intrinsic and J.E.W. supervised the project. membrane-extrinsic sectors. Reviewers: T.M.D., State University of New York Upstate Medical University; and W.D.F., Arizona State University. The membrane-extrinsic sector, known as F1-ATPase, is the catalytic part in which ATP is formed from ADP and inorganic The authors declare no conflict of interest. phosphate. It can be detached experimentally from the mem- Published under the PNAS license. brane domain in an intact state, and retains the ability to hy- Data deposition: The atomic coordinates and structure factors have been deposited in the drolyze, but not synthesize, ATP. The membrane intrinsic sector, Protein Data Bank, www.wwpdb.org (PDB ID code 6F5D). 1 sometimes called Fo, contains a rotary motor driven by pmf and M.G.M. and O.G. contributed equally to this work. is connected to the extrinsic domain by a central stalk and a 2To whom correspondence should be addressed. Email: [email protected]. peripheral stalk. The enzyme’s rotor constitutes the central stalk This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and an associated ring of c-subunits in the membrane domain. 1073/pnas.1720940115/-/DCSupplemental. The central stalk lies along an axis of sixfold pseudosymmetry Published online February 12, 2018. 2102–2107 | PNAS | February 27, 2018 | vol. 115 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1720940115 Downloaded by guest on September 24, 2021 include a hydrophobic pocket where the adenine ring of ADP (or crystallized in the presence of phosphonate (20) and in the α β ATP) is bound; a P-loop sequence that interacts with the -, -, F1-ATPase from Caldalkalibacillus thermarum (38). These struc- and γ-phosphates of ATP and provides residues involved either tures are interpreted as representing a posthydrolysis state in directly or indirectly via water molecules in the binding of a hexa- which the ADP molecule has not been released from the coordinate magnesium ion; and, in the adjacent α-subunit, an enzyme. “ ” arginine finger residue, which senses whether ADP or ATP is An unusual feature of the T. brucei F1-ATPase is that the bound to the catalytic site. Indeed, these catalytic features are diphosphate catalytic interface is more open than the tri- common to a wide range of NTPases (40, 41), and together with phosphate catalytic interface, similar to the F1-ATPase from conserved structural features are characteristic of the canonical Saccharomyces cerevisiae (24), whereas the converse is observed ATP synthase. in all other structures (Table S2). As usual, the empty interface is Based on a structural model at 32.5-Å resolution derived by the most open of the three catalytic interfaces (Table S2). The electron cryotomography (ECT), it has been suggested recently rotational position of the γ-subunit (determined by superposition that the structure of the F1-catalytic domain and its catalytic of crown regions of structures) is +23.1° relative to the bovine Trypanosoma brucei mechanism in the ATP synthase from have phosphate release dwell, which is at or close to the catalytic dwell diverged extensively from the canonical complex in an un- at +30° in the rotary catalytic cycle (6). precedented manner (42). It has been proposed that the struc- ture of this F1-domain is much more open than those described Structure of the F1-ATPase from T. brucei. The structure consists of “ ” in other species, and that the arginine finger is provided not by an α β -complex with α- and β-subunits arranged in alternation α 3 3 the -subunit, but rather by an additional p18-subunit found only around an antiparallel α-helical coiled coil in the γ-subunit (Fig. in the euglenozoa (43–49). Here we examine this proposal in γ α β T. brucei 1). The rest of the -subunit sits beneath the 3 3-complex and is the context of a structure of the F1-domain of the associated with the δ- and e-subunits. Together, these three ATP synthase determined by X-ray crystallography at 3.2- subunits form the central stalk. Thus, the overall structure of this Å resolution. catalytic domain of the ATP synthase complex is very close to Results and Discussion structures of canonical F1-ATPases determined in the mito- chondria of other species, and in eubacteria and chloroplasts. Structure Determination. The crystals of the T.