Structural And Functional Studies Into A Type Of Potential Antibiotic Drug Target NDH-2

The electron transport chain, or the respiratory chain, comprising of complexes I-V,transports the electrons derived from metabolic processes from NADH to the oxygenmolecule, and is the most important energy source for living organisms. Complex I, orNADH-ubiquinone oxidoreductase, is thus the entry point for these electrons. In manyorganisms, however, complex I could be replaced by type-II NADH dehydrogenases(NDH-2s), which catalyze the electron transfer from NADH to ubiquinone (UQ), withFAD as a prosthetic group. NDH-2s are highly conserved in many pathogenicmicroorganisms including Mycobacterium tuberculosis and Plasmodium falciparum,and have been recognized as potential drug targets. Meanwhile, heterologous expressionof Ndi1(an NDH-2in Saccharomyces cerevisiae) in human cells has been proposed totreat human diseases caused by complex I defects as typified by Parkinson’s diseases.In this study, we have solved the high-resolution crystal structures of Ndi1and itsthree types of enzyme-substrate complexes with NADH, UQ and NADH-UQ,respectively. Ndi1formed a homodimer in all the structures. Structural analysis andsequence alignment identified that Ndi1possesses a C-terminal domain (CTD) which ishighly conserved among NDH-2s. The CTD mediates the homodimerization of Ndi1,thus generating an extensive hydrophobic region in the protein to facilitate itsmembrane attachment, which has been confirmed by the subsequent biochemical andcell biological experiments. At the same time, the structures of the three Ndi1-substratecomplexes revealed the substrate binding modes in Ndi1, and demonstrated theexistence of two UQ binding sites. EPR (electron paramagnetic resonance) experimentsidentified for the first time the existence of semiquinone radicals, and the powersaturation experiments suggested that two semiquinone radicals are involved in electrontransport. Finally, a model was raised for the mechanism of electron transfer fromNADH to UQ catalyzed by Ndi1.These results laid the foundation for the studies into the detailed mechanisms ofNDH-2-mediated electron transfer, NDH-2-targeted drug designs, and the application ofNdi1in gene therapy. This study incorporated research approaches from multiple disciplines including structural biology, biochemistry, molecular biology, cell biology,and physics. The research comprehensively revealed the mechanism how Ndi1functions as an entry point for electrons to traverse the respiratory chain.