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

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.

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Figure 1: Overall structure of Na+-NQR.
Figure 2: Subunit NqrB harbours a Na+-channel.
Figure 3: Subunits NqrD and NqrE contain a novel Fe centre.
Figure 4: Subunit NqrC exhibits a new flavoprotein fold and FMN-binding motif.
Figure 5: Mechanism of redox-driven Na+ translocation.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

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).

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Acknowledgements

We thank the staff at beamlines X06SA and X06DA at Swiss Light Source for excellent support. This work was supported by contract research ‘Methoden in den Lebenswissenschaften’ of the Baden-Württemberg Stiftung P-LS-Meth/4 (to J.S. and G.F.), and by the Deutsche Forschungsgemeinschaft grant FR 1321/3-1 (to J.S.) and grant FR 1488/3-2 (to G.F.). We thank Y. Obermaier for expression and preparation of Na+-NQR.

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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.

Corresponding author

Correspondence to Günter Fritz.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 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.

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.

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.

Extended Data Figure 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.

Extended Data Figure 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.

Extended Data Figure 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.

Extended Data Figure 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.

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

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Steuber, J., Vohl, G., Casutt, M. et al. Structure of the V. cholerae Na+-pumping NADH:quinone oxidoreductase. Nature 516, 62–67 (2014). https://doi.org/10.1038/nature14003

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