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

Steuber, Julia; Vohl, Georg; Casutt, Marco S.; Vorburger, Thomas; Diederichs, Kay; Fritz, Günter

doi:10.1038/nature14003

Cited by 114 publications

(219 citation statements)

References 56 publications

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“…Recently, by alanine-scanning mutagenesis of aromatic residues in NqrB and molecular docking, Tuz et al (14) proposed that the binding site of the ubiquinone ring is located at the interface of the NqrB and NqrD subunits. The predicted site is not only inconsistent with the location of the putative cavity in NqrA (13), but is also too far from the riboflavin (ϳ36 Å) for efficient electron transfer. Because the work by Tuz et al (14) was largely based on steadystate kinetic analysis of mutants, it is difficult to determine whether the critical residues that they identified (NqrB-Phe-211 and -Phe-213) are directly involved in forming the binding pocket for the ubiquinone ring or whether they affect the reaction of ubiquinone indirectly though some long-range conformational change.…”

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confidence: 79%

“…Steuber et al (13) identified a deep solvent-accessible cavity, in the NqrA subunit, in their crystallographic structure, and pointed out that it could accommodate a ubiquinone molecule. Now, to more precisely locate the binding sites for ubiquinone and inhibitors, we have carried out photoaffinity labeling experiments with a photoreactive ubiquinone (PUQ-3) and two different aurachin-type inhibitors ([ 125 I]PAD-1 and [ 125 I]PAD-2) using the isolated Na ϩ -NQR.…”

Section: Discussionmentioning

confidence: 99%

“…1C). Although the structure of the region Met-1-Pro-37 is not included in the model of Steuber et al (13), this Ͼ200,000 (ϳ95%) Ͼ50,000 (ϳ90%) Ͼ12,000 (ϳ80%) Ͼ27,000 (ϳ100%) Ͼ10,000 (ϳ100%) NqrB-G141A 1200 (Ϯ170) 820 (Ϯ77) 3.3 (Ϯ0.14) 5200 (Ϯ610) 1.3 (Ϯ0.11) NqrB-E144C Ͼ200,000 (ϳ60%) Ͼ50,000 (ϳ70%) Ͼ12,000 (ϳ60%) Ͼ27,000 (ϳ100%) Ͼ10,000 (ϳ80%) N-terminal region would apparently form a part of the wall of the putative quinone-binding pocket identified above, because Gly-38 -Leu-53, which follows immediately in the sequence, lies in a groove neighboring the cavity in NqrA. Together, the results from the photoaffinity labeling with PUQ-3, [ (11,24,25), which showed that HQNO and korormicin do not compete directly with ubiquinone.…”

Section: Discussionmentioning

confidence: 99%

“…A recently published X-ray crystallographic study provided important information about the structure of V. cholerae Na ϩ -NQR (13), but the model includes several features that are difficult to reconcile with previous biochemical and/or biophysical functional characterizations (11). For instance, the spatial distances between several pairs of redox cofactors in the proposed electron transfer pathway are too large to support physiological rates of electron transfer; for example, the edge-toedge distance between the [2Fe-2S] cluster in NqrF and the Fe(Cys) 4 in NqrD (33.4 Å) and between the FMN and the riboflavin cofactor in NqrB (29.3 Å).…”

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confidence: 99%

“…For instance, the spatial distances between several pairs of redox cofactors in the proposed electron transfer pathway are too large to support physiological rates of electron transfer; for example, the edge-toedge distance between the [2Fe-2S] cluster in NqrF and the Fe(Cys) 4 in NqrD (33.4 Å) and between the FMN and the riboflavin cofactor in NqrB (29.3 Å). The fact that electron transfer between these cofactors takes place indicates that the subunits harboring the cofactors undergo large conformational changes during turnover that decrease these spatial gaps (13).…”

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confidence: 99%

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Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling

Ito

Murai

Ninokura

et al. 2017

Journal of Biological Chemistry

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Edited by Ruma BanerjeeThe Na ؉ -pumping NADH-quinone oxidoreductase (Na ؉ -NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae. I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na ؉ -NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.The Na ϩ -pumping NADH-quinone oxidoreductase (Na ϩ -NQR) 2 is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, such as Vibrio cholerae and Haemophilus influenzae (1, 2). Na ϩ -NQR obtains energy by oxidizing NADH and reducing ubiquinone, which allows it to generate an electrochemical Na ϩ gradient across the inner bacterial membrane. The enzyme is an integral membrane complex consisting of six subunits (NqrA-F) encoded by the nqr operon (2). There is a consensus that the electron transfer takes place through a series of five redox cofactors as follows: a FAD; a 2Fe-2S center; two covalently bound FMN, and a riboflavin (3-5). Different studies have intensively investigated the locations and redox properties of the cofactors of the enzyme (6 -12); however, the exact locations of the ubiquinone-binding site(s) and the Na ϩ transport pathway, as well as the mechanism that couples Na ϩ transport to the electron transfer, still remain elusive.A recently published X-ray crystallographic study provided important information about the structure of V. cholerae Na ϩ -NQR (13), but the model includes several features that are difficult to reconcile with previous biochemical and/or biophysical functional characterizations (11). For instance, the spatial distances between several pairs of redox cofactors in the proposed electron transfer pathway are too large to support physiological rates of electron transfer; for example, the edge-toedge distance between the [2Fe-2S] cluster in NqrF and the Fe(Cys) 4 in NqrD (33.4 Å) and between the FMN and the riboflavin cofactor in NqrB (29.3 Å). The fact that electron transfer between these cofactors takes place indicates that the subunits harboring the cofactors undergo large conformational changes during turnover that decrease these spatial gaps (13).Additionally, the crystallographic model lacks an anticipated tightly bound quinone, which has been reported in the enzyme preparations from different laboratories (4,6,8) and suggested to be located in NqrA on the basis of photoaffinity labeling and NMR studies (7,9). In the crystallographic model, the NqrA subunit includes a deep cavity that is large enough to accommodate a ubiquinone molecule, but the cavity is ϳ20 Å above the predicted membrane surface and the distance between the cavity and the riboflavin in NqrB subunit is too large (Ͼ40 Å) to be consistent with electron transfer during tu...

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confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling

Ito

Murai

Ninokura

et al. 2017

Journal of Biological Chemistry

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Structural analysis of Borrelia burgdorferi periplasmic lipoprotein BB0365 involved in Lyme disease infection

et al. 2019

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The periplasmic lipoprotein BB0365 of the Lyme disease agent Borrelia burgdorferi is expressed throughout mammalian infection and is essential for all phases of Lyme disease infection; its function, however, remains unknown. In the current study, our structural analysis of BB0365 revealed the same structural fold as that found in the NqrC and RnfG subunits of the NADH:quinone and ferredoxin:NAD+ sodium‐translocating oxidoreductase complexes, which points to a potential role for BB0365 as a component of the sodium pump. Additionally, BB0365 coordinated Zn2+ by the His51, His55, His140 residues, and the Zn2+‐binding site indicates that BB0365 could act as a potential metalloenzyme; therefore, this structure narrows down the potential functions of BB0365, an essential protein for B. burgdorferi to cause Lyme disease.

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