Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation

Abstract

SummaryNon-proton pumping type II NADH dehydrogenase (NDH-2) plays a central role in the respiratory metabolism of bacteria, and in the mitochondria of fungi, plants and protists. The lack of NDH-2 in mammalian mitochondria and its essentiality in important bacterial pathogens suggests these enzymes may represent a potential new drug target to combat microbial pathogens. Here, we report the first crystal structure of a bacterial NDH-2 enzyme at 2.5 Å resolution from Caldalkalibacillus thermarum. The NDH-2 structure reveals a homodimeric organization that has a unique dimer interface. NDH-2 is localized to the cytoplasmic membrane by two separated C-terminal membraneanchoring regions that are essential for membrane localization and FAD binding, but not NDH-2 dimerization. Comparison of bacterial NDH-2 with the yeast NADH dehydrogenase (Ndi1) structure revealed nonoverlapping binding sites for quinone and NADH in the bacterial enzyme. The bacterial NDH-2 structure establishes a framework for the structure-based design of small-molecule inhibitors.

The mechanism of catalysis by type-II NADH:quinone oxidoreductases

“…The structure of C. thermarum NDH-2 has been described, but attempts to determine the structures of its substrate-bound states were previously unsuccessful14. Here, we have determined structures of NDH-2 co-crystallized with either 1 mM NADH or 10 mM NAD + to 2.5 and 3.0 Å resolution, respectively.…”

“…Three NDH-2 homologues have been characterized structurally, from Saccharomyces cerevisiae 1213, Caldalkalibacillus thermarum 14, and Staphylococcus aureus 15. All of them are homodimers, with each monomer containing two Rossmann folds that form nucleotide-binding sites for a noncovalently bound flavin adenine dinucleotide (FAD) and the NADH substrate.…”

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)

“…These results prompted us to consider the possibility that the C-terminal polyhistidine tag might inhibit AIF association to the membrane. To explore this idea, we compared the x-ray crystal structures of mouse AIF (44) with that of Ndi1 from S. cerevisiae (45,46) and NDH-2 from Caldalkalibacillus thermarum (47). In both the Ndi1 and NDH-2 enzymes, the C-terminal regions are essential for membrane anchoring.…”

Small Molecules Targeting Mycobacterium tuberculosis Type II NADH Dehydrogenase Exhibit Antimycobacterial Activity

“…We believe that it is reasonable to suggest that the TQZ and THI scaffolds occupy the quinone and NADH binding sites, respectively, given their structural resemblance to the quinone and adenine molecules that normally reside in those positions. [20] Structural analysis coupled with kinetic enzymatic analysis of the inhibitors would be the best way to gain more insight into their inhibitory mechanism. [21] Future studies will also include utilizing our Ndh-2 inhibitors to explore the roles of Ndh-2 and Nuo in vivo, and will allow us to inactivate both or one copies of Ndh-2, respectively.…”