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PreprintCited by 3ATP Synthase and ATPases ResearchEnzyme Structure and FunctionMitochondrial Function and Pathology

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The ancestral shape of the access proton path of mitochondrial ATP synthases revealed by a split subunit-a

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Wong, Jonathan E., Zíková, Alena, Gahura, Ondřej·2023·Molecular Biology and Evolution
DOI 10.1101/2023.02.25.530031PMC PMC10306403

Preprint — published as 10.1093/molbev/msad146 · Paper page

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Abstract

The passage of protons across membranes through F 1 F o -ATP synthases spins their rotors and drives synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half-channel of mitochondrial ATP synthases are largely defined by a short N-terminal α-helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the α-helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This α-helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The α-helix blocks one of two proton routes found in Escherichia coli , resulting in the single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half-channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis.

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Fig. 1. Split subunit-a of mitochondrial ATP synthase in Trypanosoma brucei retains ancestral structure and shapes the access proton half channel. (A) Comparison of the structures of subunit-a in Saccharomyces cerevisiae and Escherichia coli and subunits a and aN in T. brucei. The c-rings are shown as a transparent surface. In the structure of S. cerevisiae, a fragment of the subunit-a from the other ATP synthase protomer is shown (su-a′). (B) The close-up view of the structures in (A). Conserved residues of hN and other selected conserved residues are shown as sticks. The residues labeled in red are directly involved in proton translocation or coordinate water molecules in the Grotthuss chain. Water molecules inferred from cryoEM density are shown as yellow balls in the T. brucei structure. (C) Structures of subunit-a of mitochondrial and bacterial ATP synthases from selected species. The structure of Rickettsia was predicted by AlphaFold.
Fig. 1. Split subunit-a of mitochondrial ATP synthase in Trypanosoma brucei retains ancestral structure and shapes the access proton half channel. (A) Comparison of the structures of subunit-a in Saccharomyces cerevisiae and Escherichia coli and subunits a and aN in T. brucei. The c-rings are shown as a transparent surface. In the structure of S. cerevisiae, a fragment of the subunit-a from the other ATP synthase protomer is shown (su-a′). (B) The close-up view of the structures in (A). Conserved residues of hN and other selected conserved residues are shown as sticks. The residues labeled in red are directly involved in proton translocation or coordinate water molecules in the Grotthuss chain. Water molecules inferred from cryoEM density are shown as yellow balls in the T. brucei structure. (C) Structures of subunit-a of mitochondrial and bacterial ATP synthases from selected species. The structure of Rickettsia was predicted by AlphaFold.CC BY-NC · PubMed Central
Fig. 2. Helix hN is conserved in most mitochondrial ATP synthases and Alphaproteobacteria. In individual lineages mapped on the phylogenetic tree based on Burki et al. (2020) and Tikhonenkov et al. (2022), the following features are shown: number of species included in the analyses (in parentheses); genome, in which subunit-a/aN is encoded (“mt”—mitochondrial, “n”—nuclear); presence or absence of helices hN and h1 of subunit-a/aN; selected representative species used in the analyses presented in the following columns (for species shown in bold a cryoEM structure of ATP synthase has been reported); sequence of hN and flanking residues (universally conserved phenylalanine is shown in red, other conserved residues of the SPLEQF motif, or the FXXF motif in Metazoa, are shown in bold); average hydrophobicity of the entire subunit-a (FL), region h1–h6, and region h5–h6 (blue—more hydrophobic, red—less hydrophobic).
Fig. 2. Helix hN is conserved in most mitochondrial ATP synthases and Alphaproteobacteria. In individual lineages mapped on the phylogenetic tree based on Burki et al. (2020) and Tikhonenkov et al. (2022), the following features are shown: number of species included in the analyses (in parentheses); genome, in which subunit-a/aN is encoded (“mt”—mitochondrial, “n”—nuclear); presence or absence of helices hN and h1 of subunit-a/aN; selected representative species used in the analyses presented in the following columns (for species shown in bold a cryoEM structure of ATP synthase has been reported); sequence of hN and flanking residues (universally conserved phenylalanine is shown in red, other conserved residues of the SPLEQF motif, or the FXXF motif in Metazoa, are shown in bold); average hydrophobicity of the entire subunit-a (FL), region h1–h6, and region h5–h6 (blue—more hydrophobic, red—less hydrophobic).CC BY-NC · PubMed Central
Fig. 3. The proton entry point and path in the access half channel are co-determined by hN. (A) Comparison of proton paths in the access half channel of the bovine mitochondrial ATP synthases with Escherichia coli and Acinetobacter baumannii counterparts. The c-ring is shown as white surface. Blue and red arrows indicate the path through the front and rear entry, respectively. (B) Superposition of the structures from E. coli and bovine mitochondria shows that hN is positionally shifted compared with the N-terminal helix of subunit-a in E. coli and that the conserved phenylalanine occupies the position of Glu219 in E. coli. Selected functionally important residues are shown as sticks in panels (A) and (B). (C) The position of the proposed front proton entry to the access half channel in A. baumannii ATP synthase (Demmer et al. 2022) is blocked by conserved phenylalanine in hN of mitochondrial and alphaproteobacterial ATP synthases. A view of the front entry (red arrowhead) to the half channel in the A. baumannii subunit-a (surface representation) from the c-ring shown individually and superposed with subunit-a from other ATP synthases (cartoon representation): Saccharomyces cerevisiae, the AlphaFold model of Rickettsia, and subunits a and aN from Trypanosoma brucei. The conserved phenylalanine is shown as sticks.
Fig. 3. The proton entry point and path in the access half channel are co-determined by hN. (A) Comparison of proton paths in the access half channel of the bovine mitochondrial ATP synthases with Escherichia coli and Acinetobacter baumannii counterparts. The c-ring is shown as white surface. Blue and red arrows indicate the path through the front and rear entry, respectively. (B) Superposition of the structures from E. coli and bovine mitochondria shows that hN is positionally shifted compared with the N-terminal helix of subunit-a in E. coli and that the conserved phenylalanine occupies the position of Glu219 in E. coli. Selected functionally important residues are shown as sticks in panels (A) and (B). (C) The position of the proposed front proton entry to the access half channel in A. baumannii ATP synthase (Demmer et al. 2022) is blocked by conserved phenylalanine in hN of mitochondrial and alphaproteobacterial ATP synthases. A view of the front entry (red arrowhead) to the half channel in the A. baumannii subunit-a (surface representation) from the c-ring shown individually and superposed with subunit-a from other ATP synthases (cartoon representation): Saccharomyces cerevisiae, the AlphaFold model of Rickettsia, and subunits a and aN from Trypanosoma brucei. The conserved phenylalanine is shown as sticks.CC BY-NC · PubMed Central

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Metadata

Authors
Wong, J. E., Zíková, A., & Gahura, O.
Affiliations
Czech Academy of Sciences (CZ)
Corresponding author
Gahura, Ondřej
ORCID
Gahura, Ondřej
Venue
Molecular Biology and Evolution
Publisher
openRxiv
Published
2023-02-26
Volume
40
Issue
6
ISSN
0737-4038 (ISSN-L)
DOI
10.1101/2023.02.25.530031
PMC
PMC10306403
OpenAlex
W4381309015
Cited by
3
References
1
Citations by year
2026: 1 · 2025: 1 · 2023: 1
FWCI
0.27
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Molecular Biology and Evolution (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • European Commission (CZ.02.1.01, HORIZON2020)
  • Ministerstvo Školství, Mládeže a Tělovýchovy (CZ.02.1.01/0.0/0.0/16_019, 0000759, 16_019/0000759, CZ.02.1.01, CZ.02.1.01/0.0/0.0/16 019/0000759)
  • Grantová Agentura České Republiky (CZ.02.1.01/0.0, GJ20-04150Y, CZ.02.1.01/0.0/0.0/ 16_019/0000759, CZ.02.1.01)
  • European Regional Development Fund (02.1.01/0.0/0.0, CZ.02, CZ.02.1.01/0.0/0.0/16_019/0000759, CZ.02.1.01, CZ.02.1.01/0.0/0.0, 16_019/0000759, 02.1.01/0.0/0.0/16_019/0000759, /16_019)
  • HORIZON EUROPE European Research Council (101044951)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › ATP Synthase and ATPases Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
Keywords
F1FO-ATP synthase, mitochondrial ATP synthase, subunit a, N-terminal alpha-helix, gene fragmentation, Trypanosoma brucei, Euglenozoa, Alphaproteobacteria, endosymbiotic origin of mitochondria, Escherichia coli
MeSH terms
Adenosine Triphosphate, Escherichia coli, Proton-Translocating ATPases, Protons, Mitochondrial Proton-Translocating ATPases, Eukaryota

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Wong, J. E., Zíková, A., & Gahura, O. (2023). The ancestral shape of the access proton path of mitochondrial ATP synthases revealed by a split subunit-a. Molecular Biology and Evolution. https://doi.org/10.1101/2023.02.25.530031

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