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In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.

ABSTRACT: We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (??)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the ? subunit, in which the C-terminal ?C fragments are displaced by ?20 Å and rotated by ?30° from their expected positions. In this location, the ?C fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert ?-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the ? and ? subunits to the fundamental process of ATP synthesis.

SUBMITTER: Muhleip AW

PROVIDER: S-EPMC5293049 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.

Mühleip Alexander W AW Dewar Caroline E CE Schnaufer Achim A Kühlbrandt Werner W Davies Karen M KM

Proceedings of the National Academy of Sciences of the United States of America 20170117 5

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F<sub>1</sub> head, in which the catalytic (αβ)<sub>3</sub> assembly forms a triangular pyramid rather than the pseudo- ...[more]

PMID: 28096380

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Project description:Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. Bacteriophage T7 gene product 4 is a model hexameric helicase that has been observed to use dTTP, but not ATP, to unwind double-stranded (ds)DNA as it translocates from 5' to 3' along single-stranded (ss)DNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective maximum rates V(max), Michaelis constants K(M) and concentrations. In contrast, processivity does not follow a simple competitive behaviour and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

Dataset Information

In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.

Publications

In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.

Similar Datasets

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