Structure of the V. cholerae Na+-pumping NADH:quinone oxidoreductase

Journal name:
Nature
Volume:
516,
Pages:
62–67
Date published:
DOI:
doi:10.1038/nature14003
Received
Accepted
Published online

Abstract

NADH oxidation in the respiratory chain is coupled to ion translocation across the membrane to build up an electrochemical gradient. The sodium-translocating NADH:quinone oxidoreductase (Na+-NQR), a membrane protein complex widespread among pathogenic bacteria, consists of six subunits, NqrA, B, C, D, E and F. To our knowledge, no structural information on the Na+-NQR complex has been available until now. Here we present the crystal structure of the Na+-NQR complex at 3.5 Å resolution. The arrangement of cofactors both at the cytoplasmic and the periplasmic side of the complex, together with a hitherto unknown iron centre in the midst of the membrane-embedded part, reveals an electron transfer pathway from the NADH-oxidizing cytoplasmic NqrF subunit across the membrane to the periplasmic NqrC, and back to the quinone reduction site on NqrA located in the cytoplasm. A sodium channel was localized in subunit NqrB, which represents the largest membrane subunit of the Na+-NQR and is structurally related to urea and ammonia transporters. On the basis of the structure we propose a mechanism of redox-driven Na+ translocation where the change in redox state of the flavin mononucleotide cofactor in NqrB triggers the transport of Na+ through the observed channel.

At a glance

Figures

  1. Overall structure of Na+-NQR.
    Figure 1: Overall structure of Na+-NQR.

    a, b, Na+-NQR is composed of six subunits, NqrA–NqrF. While NqrA (blue) contains no transmembrane helix, NqrB (orange), NqrD (magenta) and NqrE (cyan) are integral membrane proteins. NqrF (red) and NqrC (green) are anchored in the cytoplasmic membrane by a single transmembrane helix. The membrane plane is indicated by grey lines. NADH oxidation occurs at NqrF and ubiquinone (Q) has been shown to bind to NqrA. The energy liberated is used to translocate two Na+ per NADH oxidized34. b, The solvent accessible surface of Na+-NQR is shown. c, The arrangement of the redox cofactors in Na+-NQR. Edge-to-edge distances of the cofactors are indicated by broken lines.

  2. Subunit NqrB harbours a Na+-channel.
    Figure 2: Subunit NqrB harbours a Na+-channel.

    a, Analysis of the transmembrane subunits with CAVER revealed a hydrophilic channel (green) in subunit NqrB. b, Close-up view of the channel in NqrB. Transmembrane helix I is removed for clarity. The green spheres indicate the proposed path of the Na+ through NqrB. c, Key residues of the putative Na+ channel. The negatively charged side chain of Asp 346 can coordinate Na+ at the entry of the channel. The backbone carbonyl residues of Val 161, Ile 164 and Leu 168 located on transmembrane helix III can coordinate the Na+ on the predicted path. d, Ile 164, Leu 168 and on the opposite site Phe 338 and Phe 342 located on transmembrane helix VIII narrow the channel, forming a constriction.

  3. Subunits NqrD and NqrE contain a novel Fe centre.
    Figure 3: Subunits NqrD and NqrE contain a novel Fe centre.

    a, Two strong peaks in the anomalous difference map shown in orange at 5σ were detected. The first peak was assigned to the [2Fe–2S] centre in NqrF and the second peak in the centre between subunit NqrD and NqrE. The [2Fe–2S] cluster and the Fe are shown as spheres. b, c, Top view of subunits NqrD (magenta) and NqrE (cyan). NqrD and NqrE form a symmetrical dimer. Both subunits are related by a two-fold axis. The centre of this unusual dimer is formed by transmembrane helices I and IV of NqrD and NqrE, which form two half-helices instead of a continuous transmembrane helix. d, Approximately in the centre of the membrane plane the helices unfold and four Cys residues originating from each helix coordinate the Fe centre. The anomalous difference map is shown at 8σ.

  4. Subunit NqrC exhibits a new flavoprotein fold and FMN-binding motif.
    Figure 4: Subunit NqrC exhibits a new flavoprotein fold and FMN-binding motif.

    a, A part of subunit NqrD (magenta) is removed to show the localization of NqrC (green). b, The FMNNqrC is covalently bound via a phosphoester bond between the ribityl phosphate and the Thr 225 side chain. The distance between the FMN and the Fe centre in NqrD–E is just 8 Å, with the C7 and C8 methyl groups of the isoalloxazine pointing directly to the Fe centre. c, NqrC binds FMN in an unprecedented way; only the pyrimidine moiety of isoalloxazine is embedded in the protein matrix with the benzene moiety protruding from the surface. The aromatic isoalloxazine is sandwiched between Leu 145 and Leu 176. Additional hydrogen bonds by the side chains of Trp 146 and Thr 173 to N5 of FMN might stabilize its position and tune the redox potential. A further hydrogen bond between O4 of the ribityl and N1 might stabilize the radical state of the FMN.

  5. Mechanism of redox-driven Na+ translocation.
    Figure 5: Mechanism of redox-driven Na+ translocation.

    a, Model for coupling between electron transfer and Na+ translocation. NADH is oxidized at FADNqrF that mediates single electron transfer to [2Fe–2S]NqrF (i). A conformational change in NqrF is required to transfer the electron to the FeNqrD–E. This step might facilitate concomitant Na+-binding in the cytoplasmic half-channel of NqrB (ii). Subsequently the electron is transferred to FMNNqrC (iii), and FMNNqrB (iv). The latter step presumably triggers the occlusion of Na+. Electron transfer from FMNNqrB to riboflavinNqrB (RBF; v) leads to an opening of the constriction and Na+ is released to the periplasmic half channel of NqrB. The release might be dependent on the reduction of ubiquinone (vi). bd, The redox state of FMNNqrB controls the position of helix X pointing with the positive end of the helix dipole directly towards the N5. Electron transfer from FMNNqrB to riboflavinNqrB releases helix X that will move along with helix VIII. The shift of Phe 338 and Phe 342 located on helix VIII of NqrB will open the constriction and trigger translocation of the Na+ to the periplasmic half-channel. The wide periplasmic half-channel is most likely filled with water molecules, which will immediately form a water shell around the Na+.

  6. Sequence alignment of the integral membrane subunit NqrB from different organism with the corresponding subunits of the RNF complex.
    Extended Data Fig. 1: Sequence alignment of the integral membrane subunit NqrB from different organism with the corresponding subunits of the RNF complex.

    The localization of transmembrane helices is indicated by cylinders. Connecting loops located in the cytoplasm are shown in red, connecting loops located in the periplasm in blue. Thr 236 covalently binding the FMN, and Asp 346 located in the proposed Na+ channel are indicated by arrows.

  7. Sequence alignment of the integral membrane subunits NqrD and NqrE from different organism with the corresponding subunits of the RNF complex.
    Extended Data Fig. 2: Sequence alignment of the integral membrane subunits NqrD and NqrE from different organism with the corresponding subunits of the RNF complex.

    a, b, The localization of transmembrane helices is indicated by cylinders; connecting loops located in the cytoplasm are shown in red, connecting loops located in the periplasm in blue. Cys residues in NqrD (a) and NqrE (b) coordinating the Fe are indicated by arrows.

  8. Topology of the transmembrane subunits NqrB, NqrD, and NqrE and arrangement of transmembrane helices.
    Extended Data Fig. 3: Topology of the transmembrane subunits NqrB, NqrD, and NqrE and arrangement of transmembrane helices.

    ac, The schematic topology of the transmembrane helices of NqrB, NqrC and NqrD is shown on the left hand side and the corresponding structure on the right hand side. The membrane plane is indicated in grey and the cytoplasmic aspect is marked by C and the periplasmic aspect by P. a, NqrB contains ten transmembrane helices which can be divided into a N-terminal domain comprising helices I–V and a C-terminal domain comprising helices VI–X, which exhibit an inverted topology. Both domains are connected by a long periplasmic linker. The domains exhibit an inverted topology and align with an r.m.s.d. of 3.3 Å over 113 Cα positions. b, c, NqrD and NqrE each comprise six helices exhibiting an inverted topology. Helix I and helix IV of both subunits are composed of two half helices. Such an inverted topology had been predicted based on the sequence information54. d, Top view from the cytoplasmic side onto the transmembrane helices of subunits NqrB, NqrC, NqrD, NqrE and NqrF. There are a total of 24 transmembrane helices. NqrD and NqrE form a central symmetrical unit. Subunit NqrB resides on one side of the NqrD–E unit whereas the single transmembrane helices from NqrC and NqrF reside on the opposed side. NqrB is closely attached to NqrE via helices V and VI from NqrE and IV, V, IX and X from NqrB, forming an interaction surface of 1,280 Å2, whereas NqrD exhibits a much smaller contact area to NqrB via helices VI from NqrD and IV and V from NqrB, covering 335 Å2. The transmembrane helices of NqrC and NqrF are close to each other but interact with different subunits: the transmembrane helix of NqrC forms contacts with helix III of NqrD, whereas the transmembrane helix of NqrF interacts with helix III of NqrE. e, Top view of the transmembrane part of Na+-NQR and 2Fo − Fc electron density displayed at a contour level of 1.0σ. The map coefficients were sharpened by a B-factor of −80 Å2.

  9. Subunit NqrA.
    Extended Data Fig. 4: Subunit NqrA.

    a, Interactions of NqrA with other subunits in the Na+-NQR complex. The subunits of Na+-NQR are shown in different colours: NqrA in blue, NqrB in orange, NqrC in green, NqrD in magenta, NqrE in cyan, and NqrF in red. Subunit B is shown as cartoon and all other subunits as surface representation. The C-terminal domain of NqrA located proximal to the membrane forms minor contacts with the integral membrane subunit NqrB via the NqrA residues 376–379 and 425–428, located in two short loops. A long N-terminal stretch of NqrB encompassing residues 39–53 lies in a groove of NqrA interacting over a total area of 820 Å2 and anchoring NqrA to the membrane subunits. The residues shown as transparent van der Waals spheres fill almost the entire groove of NqrA. At the C terminus of NqrB, transmembrane helix 10 is elongated and protrudes into the cytoplasm, forming contacts with the C-terminal domain and the Rossmann-fold domain of NqrA, covering a total area of 430 Å2. b, c, NqrA is composed of four domains, an N-terminal domain similar to a biotin carboxyl carrier domain (blue, residues 28–100), a Rossmann-fold domain (green, residues 102–254), an ubiquitin-like domain (orange, residues 258–329), and a C-terminal helical domain (red, residues 376–446). The N-terminal residues 1–27 wrap around the Rossmann-fold domain and the ubiquitin-like domain and form two short β-strands that align with β-sheets of both domains, respectively. The C-terminal helical domain of NqrA shows similarity to a 2[4Fe–4S] cluster ferredoxin fold like for example, in fumarate reductase (PDB code 1KF6), but does not contain a FeS centre. Consistently, the Cys residues required for FeS coordination are not present in NqrA. d, Structural alignment of NqrA with Nqo1 from complex I (grey). The proteins align with an r.m.s.d. of 3.9 Å over 234 Cα positions. NqrA comprises a deep solvent-accessible cavity that is formed by residues of the Rossmann-fold domain and the ubiquitin-like domain that is large enough to accommodate ubiquinone. In case of Nqo1 of complex I the corresponding cavity harbours the isoalloxazine moiety of the FMN cofactor.

  10. A putative Na+ channel in subunit NqrB.
    Extended Data Fig. 5: A putative Na+ channel in subunit NqrB.

    a, b, Structural alignments of NqrB with urea transporter and ammonium transporter are shown. In NqrB the central helices I, III, VI and VIII form a membrane-spanning channel. Some backbone carbonyls, for example, from Val 161, Ile 164, Leu 168 from helix III deviate notably from the ideal geometry and point inwards the channel. Such a distortion indicates a putative involvement in Na+ coordination. a, The left hand side represents the side view and the right hand side the top view of NqrB (orange) aligned with bovine urea transporter (blue). Helix VIII of NqrB carrying residues forming the constriction is shown in red. The gating helices of urea transporter, which have no corresponding helices in NqrB, are depicted in dark blue. b, Structural alignment of NqrB (orange) with ammonium transporter from Archaeoglobus fulgidus. The outer helix of ammonium transporter that has no homologous helix in NqrB is shown in grey. The high structural similarity of NqrB with urea and ammonium transporter shows that the subunit preserved the basic architecture of a transporter, but has acquired an additional and completely different function as a redox protein. These structural rearrangements in the periplasmic aspect of NqrB required to embed the FMN cofactor might have contributed to the closure of the channel. c, Cross section through NqrB. The surface is coloured according to the electrostatic surface potential. The cytoplasmic half channel exhibits a negative surface charge (red) whereas the periplasmic half channel is positively charged (blue). The localization of residues Phe 338, Phe 342 and Asp 346 is indicated. The constriction is located halfway through the membrane. The borders of the cytoplasmic membrane are indicated by grey lines.

  11. Localization of riboflavin.
    Extended Data Fig. 6: Localization of riboflavin.

    A large patch of Fo − Fc density was observed between NqrB (orange) and NqrE (cyan) and assigned to the riboflavin. The isoalloxazine moiety of riboflavin fits well into the Fo − Fc density. Several interactions with the protein matrix can stabilize the riboflavin. The flavin is stacked between the side chain of Val 399 and the CB, CG of Glu 402 of NqrB on one side (Si side) and the side chain of Phe 39 of NqrE on the opposed side (Re-side). Moreover, the imidazole of His 398 of NqrB on the Si-side can form a hydrogen bond to N5 of isoalloxazine.

Tables

  1. Data collection, phasing and refinement statistics
    Extended Data Table 1: Data collection, phasing and refinement statistics
  2. Fe anomalous map peak heights
    Extended Data Table 2: Fe anomalous map peak heights
  3. Redox cofactor distances
    Extended Data Table 3: Redox cofactor distances
  4. r.m.s.d. deviations between subunits in NQR complex and the structures of the individual subunits
    Extended Data Table 4: r.m.s.d. deviations between subunits in NQR complex and the structures of the individual subunits

Accession codes

Primary accessions

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Author information

  1. These authors contributed equally to this work.

    • Georg Vohl &
    • Marco S. Casutt

Affiliations

  1. Department of Microbiology, Garbenstrasse 30, University of Hohenheim, 70599 Stuttgart, Germany

    • Julia Steuber &
    • Thomas Vorburger
  2. Institute for Neuropathology, University of Freiburg, Breisacher Strasse 64, 79106 Freiburg, Germany

    • Georg Vohl,
    • Marco S. Casutt &
    • Günter Fritz
  3. Hermann-Staudinger-Graduate school, University of Freiburg, Hebelstrasse 27, 79104 Freiburg, Germany

    • Georg Vohl
  4. Department of Biology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany

    • Kay Diederichs

Contributions

J.S., G.F. and T.V. developed expression constructs; J.S. and M.S.C. developed purification procedures; T.V., M.S.C. and G.V. expressed the protein; M.S.C., G.V. and G.F. purified the entire complex or single subunits; M.S.C., G.V. and G.F. performed crystallization, crystal harvesting and data collection. G.F. and K.D. performed data processing and determination of phases. G.F. performed model building and refinement. G.F. prepared the figures. G.F. and J.S. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Coordinates and structure factors for the entire complex of Na+-NQR and of individual subunits NqrA1–377, NqrC33–257 and NqrF129–408 have been deposited in the Protein Data Bank. The PDB accession codes are 4P6V (entire NQR complex), 4U9O (subunit NqrA, crystal 1), 4U9Q (subunit NqrA, crystal 2), 4U9S (subunit NqrC), and 4U9U (subunit NqrF).

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Sequence alignment of the integral membrane subunit NqrB from different organism with the corresponding subunits of the RNF complex. (1,022 KB)

    The localization of transmembrane helices is indicated by cylinders. Connecting loops located in the cytoplasm are shown in red, connecting loops located in the periplasm in blue. Thr 236 covalently binding the FMN, and Asp 346 located in the proposed Na+ channel are indicated by arrows.

  2. Extended Data Figure 2: Sequence alignment of the integral membrane subunits NqrD and NqrE from different organism with the corresponding subunits of the RNF complex. (1,181 KB)

    a, b, The localization of transmembrane helices is indicated by cylinders; connecting loops located in the cytoplasm are shown in red, connecting loops located in the periplasm in blue. Cys residues in NqrD (a) and NqrE (b) coordinating the Fe are indicated by arrows.

  3. Extended Data Figure 3: Topology of the transmembrane subunits NqrB, NqrD, and NqrE and arrangement of transmembrane helices. (388 KB)

    ac, The schematic topology of the transmembrane helices of NqrB, NqrC and NqrD is shown on the left hand side and the corresponding structure on the right hand side. The membrane plane is indicated in grey and the cytoplasmic aspect is marked by C and the periplasmic aspect by P. a, NqrB contains ten transmembrane helices which can be divided into a N-terminal domain comprising helices I–V and a C-terminal domain comprising helices VI–X, which exhibit an inverted topology. Both domains are connected by a long periplasmic linker. The domains exhibit an inverted topology and align with an r.m.s.d. of 3.3 Å over 113 Cα positions. b, c, NqrD and NqrE each comprise six helices exhibiting an inverted topology. Helix I and helix IV of both subunits are composed of two half helices. Such an inverted topology had been predicted based on the sequence information54. d, Top view from the cytoplasmic side onto the transmembrane helices of subunits NqrB, NqrC, NqrD, NqrE and NqrF. There are a total of 24 transmembrane helices. NqrD and NqrE form a central symmetrical unit. Subunit NqrB resides on one side of the NqrD–E unit whereas the single transmembrane helices from NqrC and NqrF reside on the opposed side. NqrB is closely attached to NqrE via helices V and VI from NqrE and IV, V, IX and X from NqrB, forming an interaction surface of 1,280 Å2, whereas NqrD exhibits a much smaller contact area to NqrB via helices VI from NqrD and IV and V from NqrB, covering 335 Å2. The transmembrane helices of NqrC and NqrF are close to each other but interact with different subunits: the transmembrane helix of NqrC forms contacts with helix III of NqrD, whereas the transmembrane helix of NqrF interacts with helix III of NqrE. e, Top view of the transmembrane part of Na+-NQR and 2Fo − Fc electron density displayed at a contour level of 1.0σ. The map coefficients were sharpened by a B-factor of −80 Å2.

  4. Extended Data Figure 4: Subunit NqrA. (526 KB)

    a, Interactions of NqrA with other subunits in the Na+-NQR complex. The subunits of Na+-NQR are shown in different colours: NqrA in blue, NqrB in orange, NqrC in green, NqrD in magenta, NqrE in cyan, and NqrF in red. Subunit B is shown as cartoon and all other subunits as surface representation. The C-terminal domain of NqrA located proximal to the membrane forms minor contacts with the integral membrane subunit NqrB via the NqrA residues 376–379 and 425–428, located in two short loops. A long N-terminal stretch of NqrB encompassing residues 39–53 lies in a groove of NqrA interacting over a total area of 820 Å2 and anchoring NqrA to the membrane subunits. The residues shown as transparent van der Waals spheres fill almost the entire groove of NqrA. At the C terminus of NqrB, transmembrane helix 10 is elongated and protrudes into the cytoplasm, forming contacts with the C-terminal domain and the Rossmann-fold domain of NqrA, covering a total area of 430 Å2. b, c, NqrA is composed of four domains, an N-terminal domain similar to a biotin carboxyl carrier domain (blue, residues 28–100), a Rossmann-fold domain (green, residues 102–254), an ubiquitin-like domain (orange, residues 258–329), and a C-terminal helical domain (red, residues 376–446). The N-terminal residues 1–27 wrap around the Rossmann-fold domain and the ubiquitin-like domain and form two short β-strands that align with β-sheets of both domains, respectively. The C-terminal helical domain of NqrA shows similarity to a 2[4Fe–4S] cluster ferredoxin fold like for example, in fumarate reductase (PDB code 1KF6), but does not contain a FeS centre. Consistently, the Cys residues required for FeS coordination are not present in NqrA. d, Structural alignment of NqrA with Nqo1 from complex I (grey). The proteins align with an r.m.s.d. of 3.9 Å over 234 Cα positions. NqrA comprises a deep solvent-accessible cavity that is formed by residues of the Rossmann-fold domain and the ubiquitin-like domain that is large enough to accommodate ubiquinone. In case of Nqo1 of complex I the corresponding cavity harbours the isoalloxazine moiety of the FMN cofactor.

  5. Extended Data Figure 5: A putative Na+ channel in subunit NqrB. (925 KB)

    a, b, Structural alignments of NqrB with urea transporter and ammonium transporter are shown. In NqrB the central helices I, III, VI and VIII form a membrane-spanning channel. Some backbone carbonyls, for example, from Val 161, Ile 164, Leu 168 from helix III deviate notably from the ideal geometry and point inwards the channel. Such a distortion indicates a putative involvement in Na+ coordination. a, The left hand side represents the side view and the right hand side the top view of NqrB (orange) aligned with bovine urea transporter (blue). Helix VIII of NqrB carrying residues forming the constriction is shown in red. The gating helices of urea transporter, which have no corresponding helices in NqrB, are depicted in dark blue. b, Structural alignment of NqrB (orange) with ammonium transporter from Archaeoglobus fulgidus. The outer helix of ammonium transporter that has no homologous helix in NqrB is shown in grey. The high structural similarity of NqrB with urea and ammonium transporter shows that the subunit preserved the basic architecture of a transporter, but has acquired an additional and completely different function as a redox protein. These structural rearrangements in the periplasmic aspect of NqrB required to embed the FMN cofactor might have contributed to the closure of the channel. c, Cross section through NqrB. The surface is coloured according to the electrostatic surface potential. The cytoplasmic half channel exhibits a negative surface charge (red) whereas the periplasmic half channel is positively charged (blue). The localization of residues Phe 338, Phe 342 and Asp 346 is indicated. The constriction is located halfway through the membrane. The borders of the cytoplasmic membrane are indicated by grey lines.

  6. Extended Data Figure 6: Localization of riboflavin. (569 KB)

    A large patch of Fo − Fc density was observed between NqrB (orange) and NqrE (cyan) and assigned to the riboflavin. The isoalloxazine moiety of riboflavin fits well into the Fo − Fc density. Several interactions with the protein matrix can stabilize the riboflavin. The flavin is stacked between the side chain of Val 399 and the CB, CG of Glu 402 of NqrB on one side (Si side) and the side chain of Phe 39 of NqrE on the opposed side (Re-side). Moreover, the imidazole of His 398 of NqrB on the Si-side can form a hydrogen bond to N5 of isoalloxazine.

Extended Data Tables

  1. Extended Data Table 1: Data collection, phasing and refinement statistics (176 KB)
  2. Extended Data Table 2: Fe anomalous map peak heights (104 KB)
  3. Extended Data Table 3: Redox cofactor distances (120 KB)
  4. Extended Data Table 4: r.m.s.d. deviations between subunits in NQR complex and the structures of the individual subunits (73 KB)

Additional data