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

Abstract

SUMMARYThe 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 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.