Ubiquinone binding site of yeast NADH dehydrogenase revealed by structures binding novel competitive- and mixed-type inhibitors.

Tetsuo Yamashita, Daniel Ken Inaoka, Tomoo Shiba, Takumi Oohashi, So Iwata, Takao Yagi, Hiroaki Kosaka, Hideto Miyoshi, Shigeharu Harada, Kiyoshi Kita, Katsuya Hirano

Journal: Scientific reports 2018;8(1):2427

PMID: 29402945

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Abstract

Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.

Address: Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Kita-gun, Kagawa, 761-0793, Japan. [email protected].; Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-0033, Japan.; School of Tropical Medicine and Global Health, Nagasaki University, 1-12-4, Sakamoto, Nagasaki, 852-8523, Japan.; Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, 606-8585, Japan.; Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London, SW7 2AZ, UK.; Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, OX11 0DE, UK.; Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Human Receptor Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.; Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-Ku, Kyoto, 606-8501, Japan.; Systems and Structural Biology Centre, RIKEN, 1-7-22 Suehiro-cho Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.; Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA.; Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Kita-gun, Kagawa, 761-0793, Japan.; Osaka Jikei College, 1-2-8 Miyahara, Yodogawa-Ku, Osaka, 532-0003, Japan.; Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.; Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, 606-8585, Japan. [email protected].

DOI: 10.1038/s41598-018-20775-6

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MeSH Terms: Amino Acid Sequence, Binding Sites, Binding, Competitive, Coenzymes, Crystallography, X-Ray, Electron Transport Complex I, Flavin-Adenine Dinucleotide, Gene Expression, Kinetics, Methacrylates, Models, Molecular, Mutation, Polyenes, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Alignment, Sequence Homology, Amino Acid, Substrate Specificity, Thermodynamics, Thiazoles, Ubiquinone