Complex I is the first enzyme of the respiratory chain and has a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation by an unknown mechanism. Dysfunction of complex I has been implicated in many human neurodegenerative diseases. We have determined the structure of its hydrophilic domain previously. Here, we report the alpha-helical structure of the membrane domain of complex I from Escherichia coli at 3.9 A resolution. The antiporter-like subunits NuoL/M/N each contain 14 conserved transmembrane (TM) helices. Two of them are discontinuous, as in some transporters. Unexpectedly, subunit NuoL also contains a 110-A long amphipathic alpha-helix, spanning almost the entire length of the domain. Furthermore, we have determined the structure of the entire complex I from Thermus thermophilus at 4.5 A resolution. The L-shaped assembly consists of the alpha-helical model for the membrane domain, with 63 TM helices, and the known structure of the hydrophilic domain. The architecture of the complex provides strong clues about the coupling mechanism: the conformational changes at the interface of the two main domains may drive the long amphipathic alpha-helix of NuoL in a piston-like motion, tilting nearby discontinuous TM helices, resulting in proton translocation.

The genes of N. pharaonis SRII and the carboxy terminal truncated transducer (1-114) were cloned into a pET27bmod expression vector 24 with a C-terminal £ 7 His tag, respectively. Proteins were expressed in Escherichia coli strain BL21 (DE3), and purified as described 25,26. After removal of imidazol by diethyl-aminoethyl chromatography, SRII-His and HtrII 114-His were mixed in a 1:1 ratio, followed by reconstitution into purple membrane (the bacteriorhodopsin containing membrane patches of H. salinarum) lipids 7 (protein to lipid ratio 1:35). After filtration, the reconstituted proteins were pelleted by centrifugation at 100,000g. For resolubilization, the samples were resuspended in a buffer containing 2% n-octyl-b-D-glucopyranoside and shaken for 16 h at 4 8C in the dark. The resolubilized complex was isolated by centrifugation at 100,000g. Crystallization, structure determination and refinement We added the solubilized complex in crystallization buffer (150 mM NaCl, 25 mM Na/KPi, pH 5.1, 0.8% n-octyl-b-D-glucopyranoside) to the lipidic phase, formed from monovaccenin (Nu-Chek Prep). Precipitant was 1 M salt Na/KPi, pH 5.6. Crystals were grown at 22 8C. X-ray diffraction data were collected at beamline ID14-1 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France, using a Quantum ADSC Q4R CCD (charge-coupled device) detector. Data were integrated using MOSFILM 27 and SCALA 28. Molecular replacement using MOLREP 28 to phase a polyalanine model (from Protein Data Bank accession number 1JGJ (ref. 12)) gave a unique solution (R ¼ 0.568, correlation coefficient C ¼ 0.357) at 2.9 A ˚. After inserting side chains for SRII, the helices of HtrII were found (R ¼ 0.329, C ¼ 0.711). Simulated annealing, positional refinement and temperature factor refinement were performed in CNS 29 ; model rebuilding was carried out in O 30 (Table 1).

Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0 Å resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.