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bioRxiv · 10.1101/2022.04.19.488743

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

Kishikawa, J.-i.·Ishikawa, M.·Masuya, T.·Murai, M.·Kitazumi, Y.·Butler, N.·Kato, T.·Barquera, B.·Miyoshi, H.
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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-[A] resolution. The structures reveal the arrangement of all six redox cofactors including riboflavin, whose position has been under debate, and a newly assigned 2Fe-2SNqrD/E cluster located between the membrane embedded NqrD and NqrE subunits. A large part of the hydrophilic NqrF near the cytoplasmic membrane surface 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, consistent with physiologically relevant electron transfer. Two different types of specific inhibitors (korormicin A and aurachin D-42) bind to the N-terminal region of NqrB, which is disordered in the absence of inhibitors. The current inhibitor-bound structures reasonably explain our previous biochemical findings obtained by different chemistry-based experiments. This study provides a definite foundation for understanding the function of Na+-NQR and the molecular mechanism of its specific inhibitors to support molecular design of new antibiotics targeting the enzyme.

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Kishikawa, J.-i., Ishikawa, M., Masuya, T., Murai, M., Kitazumi, Y., Butler, N., Kato, T., Barquera, B., Miyoshi, H.. 2022-04-19. Cryo-EM structures of Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae. https://doi.org/10.1101/2022.04.19.488743

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