A role for native lipids in the stabilization and two-dimensional crystallization of the Escherichia coli NADH-ubiquinone oxidoreductase (complex I).
NADH-ubiquinone oxidoreductase (complex I or NDH-1) was purified from the BL21 strain of Escherichia coli using an improved procedure. The complex was effectively stabilized by addition of divalent cations and lipids, making the preparation suitable for structural studies. The ubiquinone reductase activity of the enzyme was fully restored by addition of native E. coli lipids. Two different two-dimensional crystal forms, with p2 and p3 symmetry, were obtained using lipids containing native E. coli extracts. Analysis of the crystals showed that they are formed by fully intact complex I in an L-shaped conformation. Activity assays and single particle analysis indicated that complex I maintains this structure in detergent solution and does not adopt a different conformation in the active state. Thus, we provide the first experimental evidence that complex I from E. coli has an L-shape in a lipid bilayer and confirm that this is also the case for the active enzyme in solution. This suggests strongly that bacterial complex I exists in an L-shaped conformation in vivo. Our results also indicate that native lipids play an important role in the activation, stabilization and, as a consequence, crystallization of purified complex I from E. coli.
Sazanov
Leonid A
LA
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge CB2 2XY, United Kingdom. sazanov@mrc-dunn.cam.ac.uk
Carroll
Joe
J
Holt
Peter
P
Toime
Laurence
L
Fearnley
Ian M
IM
D000595:Amino Acid Sequence; D002122:Calcium Chloride / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002793:Cholic Acids; D003460:Crystallization; D019281:Dimerization; D042967:Electron Transport Complex I; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D008051:Lipid Bilayers / Q000737:chemistry; D008055:Lipids / Q000494:pharmacology*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009994:Osmolar Concentration; D011487:Protein Conformation; D012965:Sodium Chloride / Q000494:pharmacology; D012996:Solutions
D002413:Cations, Divalent; D002793:Cholic Acids; D008051:Lipid Bilayers; D008055:Lipids; D012996:Solutions; D012965:Sodium Chloride; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D002122:Calcium Chloride; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate