title: Conserved amino acid residues of the NuoD segment important for structure and function of Escherichia coli NDH-1 (complex I).
abstract: The NuoD segment (homologue of mitochondrial 49 kDa subunit) of the proton-translocating NADH:quinone oxidoreductase (complex I/NDH-1) from Escherichia coli is in the hydrophilic domain and bears many highly conserved amino acid residues. The three-dimensional structural model of NDH-1 suggests that the NuoD segment, together with the neighboring subunits, constitutes a putative quinone binding cavity. We used the homologous DNA recombination technique to clarify the role of selected key amino acid residues of the NuoD segment. Among them, residues Tyr273 and His224 were considered candidates for having important interactions with the quinone headgroup. Mutant Y273F retained partial activity but lost sensitivity to capsaicin-40. Mutant H224R scarcely affected the activity, suggesting that this residue may not be essential. His224 is located in a loop near the N-terminus of the NuoD segment (Gly217-Phe227) which is considered to form part of the quinone binding cavity. In contrast to the His224 mutation, mutants G217V, P218A, and G225V almost completely lost the activity. One region of this loop is positioned close to a cytosolic loop of the NuoA subunit in the membrane domain, and together they seem to be important in keeping the quinone binding cavity intact. The structural role of the longest helix in the NuoD segment located behind the quinone binding cavity was also investigated. Possible roles of other highly conserved residues of the NuoD segment are discussed.
authors.affiliation: Deparment of Molecular and Experimental Medicine, and ‡Department of Cell and Molecular Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, MEM256, La Jolla, California 92037, United States.
authors.forename: Prem Kumar
authors.initials: PK
authors.lastname: Sinha
authors.forename: Norma
authors.initials: N
authors.lastname: Castro-Guerrero
authors.forename: Gaurav
authors.initials: G
authors.lastname: Patki
authors.forename: Motoaki
authors.initials: M
authors.lastname: Sato
authors.forename: Jesus
authors.initials: J
authors.lastname: Torres-Bacete
authors.forename: Subhash
authors.initials: S
authors.lastname: Sinha
authors.forename: Hideto
authors.initials: H
authors.lastname: Miyoshi
authors.forename: Akemi
authors.initials: A
authors.lastname: Matsuno-Yagi
authors.forename: Takao
authors.initials: T
authors.lastname: Yagi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D017124:Conserved Sequence; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D063347:Native Polyacrylamide Gel Electrophoresis; D010088:Oxidoreductases / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; C000596778:NuoCD protein, E coli; C540656:NuoH protein, E coli; D021122:Protein Subunits; D011522:Protons; D010088:Oxidoreductases; D042967:Electron Transport Complex I