Cryo-EM structures of Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae

Abstract

The Na⁺-pumping NADH-ubiquinone oxidoreductase (Na⁺-NQR) couples electron transfer from NADH to ubiquinone with Na⁺-pumping, generating an electrochemical Na⁺ gradient that is essential for energy-consuming reactions in bacteria. Since Na⁺-NQR is exclusively found in prokaryotes, it is a promising target for highly selective antibiotics. However, the molecular mechanism of inhibition is not well-understood for lack of the atomic structural information about an inhibitor-bound state. Here we present cryo-electron microscopy structures of Na⁺-NQR from Vibrio cholerae with or without a bound inhibitor at 2.5- to 3.1-Å resolution. The structures reveal the arrangement of all six redox cofactors including a herein identified 2Fe-2S cluster located between the NqrD and NqrE subunits. A large part of the hydrophilic NqrF is barely visible in the density map, suggesting a high degree of flexibility. This flexibility may be responsible to reducing the long distance between the 2Fe-2S centers in NqrF and NqrD/E. Two different types of specific inhibitors bind to the N-terminal region of NqrB, which is disordered in the absence of inhibitors. The present study provides a foundation for understanding the function of Na⁺-NQR and the binding manner of specific inhibitors.

Fig. 1. Overall structure of V. cholerae Na⁺-NQR.

The density map A and the atomic model B of Na⁺-NQR, showing the six subunits NqrA–F. The density map and the atomic model are represented as semi-transparent surface and cartoon, respectively. The hydrophilic NqrA (blue) contains no TMH. NqrC (pale blue) and NqrF (red) are hydrophilic proteins, each anchored by a single TMH. The NqrB (yellow), NqrD (pink), and NqrE (green) subunits are integral membrane proteins. The membrane plane is indicated by gray lines. C Overlay of cryo-EM structure of the membrane domain (color) with the crystallographic structure (gray, 16). NqrB, NqrC, NqrD, NqrE, and NqrF subunits have 10, 1, 6, 6, and 1 TMHs, respectively. TMHs are shown as cylinder models. The membrane domain contains a total of 24 TMHs. In the leftmost inset, the membrane interior loop (Gly266–Ser276) in the cryo-EM structure is indicated in red. The hydrophilic domain of each subunit, which protrudes from the membrane, has been deleted for clarity. TMHs are numbered.

Fig. 2. The cytoplasmic contact area between NqrA and NqrF.

NqrF binds tightly to the neighboring hydrophilic NqrA via electrostatic interactions in the state 1 model. Lower panels show cross-section images of the contact area between the two subunits. The positively and negatively charged residues in the NqrA and NqrF are indicated in blue and red, respectively.

Fig. 3. The putative 2Fe-2S center located between subunits NqrD and NqrE that is so far undetected.

The density map at the interface between NqrD and NqrE resembles that of a 2Fe-2S cluster rather than a single iron atom. An upper panel: The 2Fe-2S cluster at the center of the two-fold symmetrical axis of NqrD and NqrE. A lower panel: The two pairs of conserved cysteine residues (NqrD-Cys29/NqrE-Cys120 and NqrE-Cys26 and NqrD-Cys112) coordinate the 2Fe-2S cluster. The Cys residues and 2Fe-2S cluster are shown as sticks and ball-and-sticks models, respectively. In this panel, the consensus map for Na⁺-NQR is used and shown as a mesh representation.

Fig. 4. Riboflavin is located in NqrB.

A, B The positions of riboflavin (RBF) and FMN in NqrB are indicated. Riboflavin (sticks model) is surrounded by central TMHs 1, 3, 5, and 8 (cartoon model). C Close-up view of the binding site of riboflavin (ball-and-sticks model). The density map is shown as a mesh representation. The residues suggested to be involved in hydrogen bonds with riboflavin are indicated.

Fig. 5. Positions of all cofactors in Na⁺-NQR.

A The structure around FMN in NqrC. The head-group of FMN protrudes from the NqrC protein matrix and lies in the periplasmic medium cavity formed by NqrB, NqrD, and NqrE (an upper panel). FMN^NqrC and FMN^NqrB face each other in the closed pocket at the periplasmic interface between NqrB and NqrC. The residues that interact with FMN^NqrC and FMN^NqrB are indicated (a lower panel). The possible hydrogen bonds are represented as dashed lines. The positions of all cofactors in the present cryo-EM structure (B) and the X-ray crystallographic structure (C) are illustrated. The edge-to-edge distances of the cofactors are indicated.

Fig. 6. Structures of Na⁺-NQR with bound inhibitors.

Aurachin D-42 (A) and korormicin A (B) bind to the N-terminal region starting with TMH 1 of NqrB. Left and right panels show the overall and enlarged views of the binding pockets, respectively. The residues that are involved in the interaction with the inhibitors are indicated as sticks model. The density maps are shown as mesh representations. The disordered region (NqrB-Gly2–Leu26) in the absence of an inhibitor is indicated in dark brown (a left panel). The possible hydrogen bonds are represented as dashed lines. Water molecules are shown as red spheres.