ATP synthase from Trypanosoma brucei has an elaborated canonical F ₁ -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 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 F-ATPases determined previously. The αβ-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 F-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.

Structure‐Based Design, Synthesis and Biological Evaluation of Bis‐Tetrahydropyran Furan Acetogenin Mimics Targeting the Trypanosomatid F1 Component of ATP Synthase

“…To confirm that AutoDock Vina had been set up correctly, and to estimate binding affinity in kcal/mol, ADP and ATP were docked into the catalytic site (corresponding to the ATP‐bound conformation) of F1 from T. brucei , . Both compounds docked suitably close to the position of ADP detected within the F1 crystal structure (Figure A–B), although the β‐phosphate was positioned where the γ‐phosphate of ATP would normally reside, rather than in its own pocket.…”

“…This correlation between loss of activity against F1 and against cells by furans 10–12 implies that in these cells, compounds 4–9 induce their cytotoxic activities through F1 inhibition. Given that mammalian and T. brucei F1 are highly similar, and that 4–9 are relatively non‐specific towards trypanosomatid cells, the design of trypanosomatid‐specific F1 inhibitors of this type may prove challenging.…”

“…Protein receptor sites – the F1 crystal structures from T. brucei (6F5D.pdb) was used for docking with Autodock Vina . The receptor was prepared exactly as described in the user manual using Autodock tools .…”

“…Such bis‐tetrahydropyran 2,5‐substituted furan compounds share a degree of structural similarity with DB75, a bis‐phenyl 2,5‐furan diamidine, which is also reported to inhibit the trypanosomatid FoF1‐ATP synthase . Herein we undertook computational docking of furans into the recently disclosed crystal structure of F1 from T. brucei to identify compounds with potential trypanocidal activities. We then undertook the synthesis of a targeted library of stereochemically complex furan derivatives identified as potential enzyme binders and assessed their biological potential in cytotoxicity assays and F1 assays to confirm their on‐target activities.…”

The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans

“…Physical interaction of T. brucei ATP synthase subunit c with TbMCU. As a "reverse" approach to identify the association of TbMCU with a potential ATP synthasome, we generated in situ HA-tagged T. brucei ATP␤ (TbATP␤), TbATPp18 (a subunit that binds to each of the TbATP␣ subunits [44,45]), TbANT, and TbPiC PCF cell lines. These proteins colocalized with MitoTracker (MT) to mitochondria of T. brucei PCF (Fig.…”