Projection structure of the membrane domain of Escherichia coli respiratory complex I at 8 A resolution

Respiratory complex I (NADH:ubiquinone oxidoreductase) is an L-shaped multisubunit protein assembly consisting of a hydrophobic membrane arm and a hydrophilic peripheral arm. It catalyses the transfer of two electrons from NADH to quinone coupled to the translocation of four protons across the membrane. Although we have solved recently the crystal structure of the peripheral arm, the structure of the complete enzyme and the coupling mechanism are not yet known. The membrane domain of Escherichia coli complex I consists of seven different subunits with total molecular mass of 258 kDa. It is significantly more stable than the whole enzyme, which allowed us to obtain well-ordered two-dimensional crystals of the domain, belonging to the space group p22(1)2(1). Comparison of the projection map of negatively stained crystals with previously published low-resolution structures indicated that the characteristic curved shape of the membrane domain is remarkably well conserved between bacterial and mitochondrial enzymes, helping us to interpret projection maps in the context of the intact complex. Two pronounced stain-excluding densities at the distal end of the membrane domain are likely to represent the two large antiporter-like subunits NuoL and NuoM. Cryo-electron microscopy on frozen-hydrated crystals allowed us to calculate a projection map at 8 A resolution. About 60 transmembrane alpha-helices, both perpendicular to the membrane plane and tilted, are present within one membrane domain, which is consistent with secondary structure predictions. A possible binding site and access channel for quinone are found at the interface with the peripheral arm. Tentative assignment of individual subunits to the features of the map has been made. The location of subunits NuoL and NuoM at substantial distance from the peripheral arm, which contains all the redox centres of the complex, indicates that conformational changes are likely to play a role in the mechanism of coupling between electron transfer and proton pumping.     

Three-dimensional structure of the large ribosomal subunit from Escherichia coli.

The three-dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.     

Three-dimensional structure of the KdpFABC complex of Escherichia coli by electron tomography of two-dimensional crystals

The KdpFABC complex (Kdp) functions as a K⁺ pump in Escherichia coli and is a member of the family of P-type ATPases. Unlike other family members, Kdp has a unique oligomeric composition and is notable for segregating K⁺ transport and ATP hydrolysis onto separate subunits (KdpA and KdpB, respectively). We have produced two-dimensional crystals of the KdpFABC complex within reconstituted lipid bilayers and determined its three-dimensional structure from negatively stained samples using a combination of electron tomography and real-space averaging. The resulting map is at a resolution of 2.4 nm and reveals a dimer of Kdp molecules as the asymmetric unit; however, only the cytoplasmic domains are visible due to the lack of stain penetration within the lipid bilayer. The sizes of these cytoplasmic domains are consistent with Kdp and, using a pseudo-atomic model, we have described the subunit interactions that stabilize the Kdp dimer within the larger crystallographic array. These results illustrate the utility of electron tomography in structure determination of ordered assemblies, especially when disorder is severe enough to hamper conventional crystallographic analysis.     

Three-dimensional structure of 4-amino-4-deoxychorismate lyase from Escherichia coli

4-Amino-4-deoxychorismate lyase (ADCL) is a member of the fold-type IV of PLP dependent enzymes that converts 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate and pyruvate. The crystal structure of ADCL from Escherichia coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 20.6% at 2.2 A resolution. The enzyme is a homo dimer with a crystallographic twofold axis, and the polypeptide chain is folded into small and large domains with an interdomain loop. The coenzyme, pyridoxal 5'-phosphate, resides at the domain interface, its re-face facing toward the protein. Although the main chain folding of the active site is homologous to those of D-amino acid and L-branched-chain amino acid aminotransferases, no residues in the active site are conserved among them except for Arg59, Lys159, and Glu193, which directly interact with the coenzyme and play critical roles in the catalytic functions. ADC was modeled into the active site of the unliganded enzyme on the basis of the X-ray structures of the unliganded and liganded forms in the D-amino acid and L-branched-chain amino acid aminotransferases. According to this model, the carboxylates of ADC are recognized by Asn256, Arg107, and Lys97, and the cyclohexadiene moiety makes van der Waals contact with the side chain of Leu258. ADC forms a Schiff base with PLP to release the catalytic residue Lys159, which forms a hydrogen bond with Thr38. The neutral amino group of Lys159 eliminates the a-proton of ADC to give a quinonoid intermediate to release a pyruvate in accord with the proton transfer from Thr38 to the olefin moiety of ADC.     

Molecular structure of exonuclease I from Escherichia coli B

Exonuclease I of Escherichia coli B (EC 3.1.4.25) was determined to be a monomeric protein of molecular weight approx. 72,000, as estimated by Sephadex gel filtration, sedimentation velocity centrifugation, and sodium dodecylsulfate polyacrylamide gel electrophoresis.     

Tightly-bound ubiquinone in the Escherichia coli respiratory Complex I

NADH:ubiquinone oxidoreductase (Complex I), the electron input enzyme in the respiratory chain of mitochondria and many bacteria, couples electron transport to proton translocation across the membrane. Complex I is a primary proton pump; although its proton translocation mechanism is yet to be known, it is considered radically different from any other mechanism known for redox-driven proton pumps: no redox centers have been found in its membrane domain where the proton translocation takes place. Here we studied the properties and the catalytic role of the enzyme-bound ubiquinone in the solubilized, purified Complex I from Escherichia coli. The ubiquinone content in the enzyme preparations was 1.3±0.1 per bound FMN residue. Rapid mixing of Complex I with NADH, traced optically, demonstrated that both reduction and re-oxidation kinetics of ubiquinone coincide with the respective kinetics of the majority of Fe-S clusters, indicating kinetic competence of the detected ubiquinone. Optical spectroelectrochemical redox titration of Complex I followed at 270-280nm, where the redox changes of ubiquinone contribute, did not reveal any transition within the redox potential range typical for the membrane pool, or loosely bound ubiquinone (ca. +50-+100mV vs. NHE, pH 6.8). The transition is likely to take place at much lower potentials (E(m) ≤-200mV). Such perturbed redox properties of ubiquinone indicate that it is tightly bound to the enzyme's hydrophobic core. The possibility of two ubiquinone-binding sites in Complex I is discussed.     

Three-dimensional structure of AzoR from Escherichia coli. An oxidereductase conserved in microorganisms

The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2 A resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR.     

Three-dimensional structure of aspartate aminotransferase from Escherichia coli at 2.8 A resolution

The crystal structure of aspartate aminotransferase of Escherichia coli was determined by X-ray structure analysis at 2.8 A resolution. The structure was solved by the molecular replacement method and refined to an R-factor of 0.27, and it was found that the overall structure of AspAT of E. coli is similar to that of those of higher animals.     

Three-dimensional structure of thymidine phosphorylase from Escherichia coli at 2.8 A resolution

The three-dimensional structure of thymidine phosphorylase from Escherichia coli has been determined at 2.8 A resolution using multiple-isomorphous-replacement techniques. The amino acid sequence deduced from the deoA DNA sequence is also reported. Thymidine phosphorylase exists in the crystal as an S-shaped dimer in which the subunits are related by a crystallographic 2-fold axis. Each subunit is composed of a small alpha-helical domain of six helices and a large alpha/beta domain. The alpha/beta domain includes a six-stranded mixed beta-sheet and a four-stranded antiparallel beta-sheet. The active site has been identified by difference Fourier analyses of the binding of thymine and thymidine and lies in a cavity between the small and large domains. The central beta-sheet is splayed open to accommodate a putative phosphate-binding site which is probably occupied by a sulfate ion in the crystal.     

Three-dimensional structure prediction of the NAD binding site of proton-pumping transhydrogenase from Escherichia coli

A three-dimensional structure of the NAD site of Escerichia coli transhydrogenase has been predicted. The model is based on analysis of conserved residues among the transhydrogenases from five different sources, homologies with enzymes using NAD as cofactors or substrates, hydrophilicity profiles, and secondary structure predictions. The present model supports the hypothesis that there is one binding site, located relatively close to the N-terminus of the α-subunit. The proposed structure spans residues α145 to α287, and it includes five β-strands and five α-helices oriented in a typical open twisted α/β conformation. The amino acid sequence following the GXGXXG dinucleotide binding consensus sequence (residues α172 to α177) correlates exactly to a typical fingerprint region for ADP binding βαβ folds in dinucleotide binding enzymes. In the model, aspartic acid α195 forms hydrogen bonds to one or both hydroxyl groups on the adenosine ribose sugar moiety. Threonine α196 and alanine α256, located at the end of βB and βD, respectively, create a hydrophobic sandwich with the adenine part of NAD buried inside. The nicotinamide part is located in a hydrophobic cleft between αA and βE. Mutagenesis work has been carried out in order to test the predicted model and to determine whether residues within this domain are important for proton pumping directly. All data support the predicted structure, and no residue crucial for proton pumping Was detected. Since no three-dimensional structure of transhydrogenase has been solved, a well based tertiary structure prediction is of great value for further experimental design in trying to elucidate the mechanism of the energy-linked proton pump. © 1995 Wiley-Liss, Inc.     

The cryo-EM structure of a translation initiation complex from Escherichia coli

The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex (IC) stalled after 70S assembly. We have obtained a cryo-EM reconstruction of the IC showing IF2*GDPNP at the intersubunit cleft of the 70S ribosome. IF2*GDPNP contacts the 30S and 50S subunits as well as fMet-tRNA(fMet). IF2 here adopts a conformation radically different from that seen in the recent crystal structure of IF2. The C-terminal domain of IF2 binds to the single-stranded portion of fMet-tRNA(fMet), thereby forcing the tRNA into a novel orientation at the P site. The GTP binding domain of IF2 binds to the GTPase-associated center of the 50S subunit in a manner similar to EF-G and EF-Tu. Additionally, we present evidence for the localization of IF1, IF3, one C-terminal domain of L7/L12, and the N-terminal domain of IF2 in the initiation complex.     

Three-dimensional reconstruction of the ribosome from Escherichia coli.

Three-dimensional image reconstruction has been applied to electron micrographs of noncrystalline, negatively stained ribosomes obtained from Escherichia coli. Several independent reconstructions all show an overall appearance resembling models that had been derived earlier by direct visual interpretation of electron micrographs. The reconstructed ribosomes show numerous structural details not recognized previously, some of which may be functionally significant. A large elongate cavity (approximately 8-nm long x 5-nm wide x 6-nm [maximal] deep) is present on the surface of the ribosome near the base of its stalk and is identifiable as a portion of a feature termed the interface canyon, which was detected in prior reconstructions of the large ribosomal subunit (Radermacher, M., T. Wagenknecht, A. Verschoor, and J. Frank. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1107-1114). On the back of the ribosome, near the base of the central protuberance, is a hole leading to the interface canyon, which likely represents an exit site for the elongating polypeptide produced during protein biosynthesis. The exposed portion of the interface canyon appears well suited to bind two tRNA molecules in a configuration that is consistent with biochemical and structural data on the mechanism of peptide bond biosynthesis.     

Binding of nucleotides to the ATP-dependent protease La from Escherichia coli

A critical enzyme in protein breakdown in Escherichia coli is the ATP-hydrolyzing protease La, the lon gene product. In order to clarify the role of ATP in proteolysis, we studied ATP and ADP binding to this enzyme using rapid gel filtration to separate free from bound ligands. In the presence of Mg2+ or Mn2+ and 10 microM ATP, two molecules of ATP were bound to the tetrameric enzyme, while at 100 microM ATP (or higher), four ATP molecules were bound, both at 0 and 37 degrees C. Protease La thus has two high affinity sites (S0.5 less than 10(-7) M) for ATP and two lower affinity sites (S0.5 = 12-15 microM). Binding was reversible. In the absence of a divalent ion, ATP bound to only two sites. However, much lower Mg2+ concentrations (50 microM) were required for maximal ATPase binding than for maximal proteolytic and ATPase activity (2 mM). Decavanadate, which is a potent inhibitor of proteolysis, also blocked ATP binding, but orthovanadate had neither effect. Different ATP analogs bind to these sites in distinct ways. Adenyl-5'-yl imidodiphosphate binds to only one high affinity site, while adenyl-5'-yl methylene monophosphonate binds to two. Nevertheless, both non-metabolizable analogs can activate oligopeptide hydrolysis as well as ATP. Although binding of a single nucleotide can activate peptide hydrolysis, occupancy of all four sites appears necessary for maximal protein breakdown. The ATP molecules on all four sites are hydrolyzed rapidly. The Pi is released, but ADP remains on the enzyme. ADP binds to the same four sites, but this process does not require divalent ions. Protease La shows higher affinity for ADP than for ATP. Therefore, in vivo, ADP should inhibit ATP binding and protease La function.     

Three-dimensional reconstruction of the 70S Escherichia coli ribosome in ice: the distribution of ribosomal RNA

A reconstruction, at 40 A, of the Escherichia coli ribosome imaged by cryo-electron microscopy, obtained from 303 projections by a single-particle method of reconstruction, shows the two subunits with unprecedented clarity. In the interior of the subunits, a complex distribution of higher mass density is recognized, which is attributed to ribosomal RNA. The masses corresponding to the 16S and 23S components are linked in the region of the platform of the small subunit. Thus the topography of the rRNA regions responsible for protein synthesis can be described.