New crystal forms of the integral membrane Escherichia coli quinol:fumarate reductase suggest that ligands control domain movement.

Quinol:fumarate reductase (QFR) is an integral membrane protein and a member of the respiratory Complex II superfamily. Although the structure of Escherichia coli QFR was first reported almost twenty years ago, many open questions of catalysis remain. Here we report two new crystal forms of QFR, one grown from the lipidic cubic phase and one grown from dodecyl maltoside micelles. QFR crystals grown from the lipid cubic phase processed as P1, merged to 7.5 Šresolution, and exhibited crystal packing similar to previous crystal forms. Crystals grown from dodecyl maltoside micelles processed as P21, merged to 3.35 Šresolution, and displayed a unique crystal packing. This latter crystal form provides the first view of the E. coli QFR active site without a dicarboxylate ligand. Instead, an unidentified anion binds at a shifted position. In one of the molecules in the asymmetric unit, this is accompanied by rotation of the capping domain of the catalytic subunit. In the other molecule, this is associated with loss of interpretable electron density for this same capping domain. Analysis of the structure suggests that the ligand adjusts the position of the capping domain.

A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.

GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB(BR)), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB(BR) structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspB(BR)-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspB(BR). This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase.

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.

Structure of Escherichia coli succinate:quinone oxidoreductase with an occupied and empty quinone-binding site.

Three new structures of Escherichia coli succinate-quinone oxidoreductase (SQR) have been solved. One with the specific quinone-binding site (Q-site) inhibitor carboxin present has been solved at 2.4 A resolution and reveals how carboxin inhibits the Q-site. The other new structures are with the Q-site inhibitor pentachlorophenol and with an empty Q-site. These structures reveal important details unresolved in earlier structures. Comparison of the new SQR structures shows how subtle rearrangements of the quinone-binding site accommodate the different inhibitors. The position of conserved water molecules near the quinone binding pocket leads to a reassessment of possible water-mediated proton uptake networks that complete reduction of ubiquinone. The dicarboxylate-binding site in the soluble domain of SQR is highly similar to that seen in high resolution structures of avian SQR (PDB 2H88) and soluble flavocytochrome c (PDB 1QJD) showing mechanistically significant structural features conserved across prokaryotic and eukaryotic SQRs.

Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB.

The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspB(BR)) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspB(BR) buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspB(BR) in each buffer. While both sets of conditions supported crystal growth in space group P2(1)2(1)2(1), the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 Šfor crystal form 1 and a = 34.6, b = 98.3, c = 99.0 Šfor crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 Šresolution. A complete data set has been collected to 1.3 Šresolution with an overall R(merge) value of 0.04 and an R(merge) value of 0.33 in the highest resolution shell.

Purification, crystallisation and preliminary crystallographic studies of succinate:ubiquinone oxidoreductase from Escherichia coli.

A membrane protein complex, succinate dehydrogenase (SQR) from Escherichia coli has been purified and crystallised. This enzyme is composed of four subunits containing FAD, three iron-sulphur clusters and one haem b as prosthetic groups. The obtained crystals belong to the hexagonal space group P6(3) with the unit-cell dimensions of a=b=123.8 A and c=214.6 A. An asymmetric unit of the crystals contains one SQR monomer (M(r) 120 kDa). A data set is now available at 4.0 A resolution with 88.1% completeness and 0.106 R(merge). We have obtained a molecular replacement solution that shows sensible molecular packing, using the soluble domain of E. coli QFR (fumarate reductase) as a search model. The packing suggests that E. coli SQR is a crystallographic trimer rather than a dimer as observed for the E. coli QFR.

Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases.

The anaerobically expressed fumarate reductase and aerobically expressed succinate dehydrogenase from Escherichia coli comprise two different classes of succinate:quinone oxidoreductases (SQR), often termed respiratory complex II. The X-ray structures of both membrane-bound complexes have revealed that while the catalytic/soluble domains are structurally similar the quinone binding domains of the enzyme complexes are significantly different. These results suggest that the anaerobic and aerobic forms of complex II have evolved different mechanisms for electron and proton transfer in their respective membrane domains.

Structural and computational analysis of the quinone-binding site of complex II (succinate-ubiquinone oxidoreductase): a mechanism of electron transfer and proton conduction during ubiquinone reduction.

The transfer of electrons and protons between membrane-bound respiratory complexes is facilitated by lipid-soluble redox-active quinone molecules (Q). This work presents a structural analysis of the quinone-binding site (Q-site) identified in succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli. SQR, often referred to as Complex II or succinate dehydrogenase, is a functional member of the Krebs cycle and the aerobic respiratory chain and couples the oxidation of succinate to fumarate with the reduction of quinone to quinol (QH(2)). The interaction between ubiquinone and the Q-site of the protein appears to be mediated solely by hydrogen bonding between the O1 carbonyl group of the quinone and the side chain of a conserved tyrosine residue. In this work, SQR was co-crystallized with the ubiquinone binding-site inhibitor Atpenin A5 (AA5) to confirm the binding position of the inhibitor and reveal additional structural details of the Q-site. The electron density for AA5 was located within the same hydrophobic pocket as ubiquinone at, however, a different position within the pocket. AA5 was bound deeper into the site prompting further assessment using protein-ligand docking experiments in silico. The initial interpretation of the Q-site was re-evaluated in the light of the new SQR-AA5 structure and protein-ligand docking data. Two binding positions, the Q(1)-site and Q(2)-site, are proposed for the E. coli SQR quinone-binding site to explain these data. At the Q(2)-site, the side chains of a serine and histidine residue are suitably positioned to provide hydrogen bonding partners to the O4 carbonyl and methoxy groups of ubiquinone, respectively. This allows us to propose a mechanism for the reduction of ubiquinone during the catalytic turnover of the enzyme.

Cloning, sequencing, and heterologous expression of the murine peroxisomal flavoprotein, N1-acetylated polyamine oxidase.

The aminoacyl sequences of three regions of pure bovine N1-acetylated polyamine oxidase (PAO) were obtained and used to search GenBankTM. This led to the cloning and sequencing of a complete coding cDNA for murine PAO (mPAO) and the 5'-truncated coding region of the bovine pao (bpao) gene. A search of GenBankTM indicated that mpao maps to murine chromosome 7 as seven exons. The translated amino acid sequences of mpao and bpao have a -Pro-Arg-Leu peroxisomal targeting signal at the extreme C termini. A beta-alpha-beta FAD-binding motif is present in the N-terminal portion of mPAO. This and several other regions of mPAO and bPAO are highly similar to corresponding sections of other flavoprotein amine oxidases, although the overall identity of aligned sequences indicates that PAO represents a new subfamily of flavoproteins. A fragment of mpao was used as a probe to establish the relative transcription levels of this gene in various mature murine tissues and murine embryonic and breast tissues at different developmental stages. An Escherichia coli expression system has been developed for manufacturing mPAO at a reasonable level. The mPAO so produced was purified to homogeneity and characterized. It was demonstrated definitively that PAO oxidizes N1-acetylspermine to spermidine and 3-acetamidopropanal and that it also oxidizes N1-acetylspermidine to putrescine and 3-acetamidopropanal. Thus, this is the classical polyamine oxidase (EC 1.5.3.11) that is defined as the enzyme that oxidizes these N1-acetylated polyamines on the exo-side of their N4-amino groups. This enzyme is distinguishable from the plant polyamine oxidase that oxidizes spermine on the endo-side of the N4-nitrogen. It differs also from mammalian spermine oxidase that oxidizes spermine (but not N1-acetylspermine or N1-acetylspermidine) at the exo-carbon of its N4-amino group. This report provides details of the biochemical, spectral, oxidation-reduction, and steady-state kinetic properties of pure mPAO.

Using rational screening and electron microscopy to optimize the crystallization of succinate:ubiquinone oxidoreductase from Escherichia coli.

The membrane-bound respiratory complex II, succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli, has been anaerobically expressed, then purified and crystallized. The initial crystals obtained were small and diffracted poorly. In order to facilitate structure determination, rational screening and sample-quality analysis using electron microscopy was implemented. The crystals of SQR from E. coli belong to the trigonal space group R32, with unit-cell parameters a = b = 138.7, c = 521.9 A, and diffract to 2.6 A resolution. The optimization strategy used for obtaining well diffracting SQR crystals is applicable to a wide range of membrane proteins.

Architecture of succinate dehydrogenase and reactive oxygen species generation.

The structure of Escherichia coli succinate dehydrogenase (SQR), analogous to the mitochondrial respiratory complex II, has been determined, revealing the electron transport pathway from the electron donor, succinate, to the terminal electron acceptor, ubiquinone. It was found that the SQR redox centers are arranged in a manner that aids the prevention of reactive oxygen species (ROS) formation at the flavin adenine dinucleotide. This is likely to be the main reason SQR is expressed during aerobic respiration rather than the related enzyme fumarate reductase, which produces high levels of ROS. Furthermore, symptoms of genetic disorders associated with mitochondrial SQR mutations may be a result of ROS formation resulting from impaired electron transport in the enzyme.