Search tips
Search criteria

Results 1-9 (9)

Clipboard (0)

Select a Filter Below

Year of Publication
Document Types
1.  Structural basis of interprotein electron transfer in bacterial sulfite oxidation 
eLife  null;4:e09066.
Interprotein electron transfer underpins the essential processes of life and relies on the formation of specific, yet transient protein-protein interactions. In biological systems, the detoxification of sulfite is catalyzed by the sulfite-oxidizing enzymes (SOEs), which interact with an electron acceptor for catalytic turnover. Here, we report the structural and functional analyses of the SOE SorT from Sinorhizobium meliloti and its cognate electron acceptor SorU. Kinetic and thermodynamic analyses of the SorT/SorU interaction show the complex is dynamic in solution, and that the proteins interact with Kd = 13.5 ± 0.8 μM. The crystal structures of the oxidized SorT and SorU, both in isolation and in complex, reveal the interface to be remarkably electrostatic, with an unusually large number of direct hydrogen bonding interactions. The assembly of the complex is accompanied by an adjustment in the structure of SorU, and conformational sampling provides a mechanism for dissociation of the SorT/SorU assembly.
eLife digest
A key feature of many important chemical reactions in cells is the transfer of particles called electrons from one molecule to another. The sulfite oxidizing enzymes (or SOEs) are a group of enzymes that are found in many organisms. These enzymes convert sulfite, which is a very reactive compound that can damage cells, into another compound called sulfate. As part of this process the SOE transfers electrons from sulfite to other molecules, such as oxygen or a protein called cytochrome c. In the past, researchers have described the three-dimensional structure of three SOEs using a technique called X-ray crystallography. However, it has been difficult to study how SOEs pass electrons to other molecules because of the temporary nature of the interactions.
McGrath et al. studied an SOE called SorT, which is found in bacteria. The SorT enzyme passes electrons from sulfite to another protein called SorU. McGrath used X-ray crystallography to determine the three-dimensional structures of versions of these proteins from a bacterium called Sinorhizobium meliloti. This included structures of the proteins on their own, and when they were bound to each other. These structures revealed that a subtle change in the shape of SorU occurs when the proteins interact, which enables an electron to be quickly transferred.
McGrath et al. also found that the interface between the two proteins showed an unexpectedly high number of contact sites. These strengthen the interaction between the two proteins, which helps to make electron transfer more efficient. However, these contact sites do not prevent the two proteins from quickly moving apart after the electrons have been transferred. The next challenge is to find out whether these observations are common to SOEs from other forms of life.
PMCID: PMC4760952  PMID: 26687009
sinorhizobium meliloti; electron transfer; structural biology; molybdenum; sulfite oxidase; Other
2.  Dysregulation of transition metal ion homeostasis is the molecular basis for cadmium toxicity in Streptococcus pneumoniae 
Nature Communications  2015;6:6418.
Cadmium is a transition metal ion that is highly toxic in biological systems. Although relatively rare in the Earth’s crust, anthropogenic release of cadmium since industrialization has increased biogeochemical cycling and the abundance of the ion in the biosphere. Despite this, the molecular basis of its toxicity remains unclear. Here we combine metal-accumulation assays, high-resolution structural data and biochemical analyses to show that cadmium toxicity, in Streptococcus pneumoniae, occurs via perturbation of first row transition metal ion homeostasis. We show that cadmium uptake reduces the millimolar cellular accumulation of manganese and zinc, and thereby increases sensitivity to oxidative stress. Despite this, high cellular concentrations of cadmium (~17 mM) are tolerated, with negligible impact on growth or sensitivity to oxidative stress, when manganese and glutathione are abundant. Collectively, this work provides insight into the molecular basis of cadmium toxicity in prokaryotes, and the connection between cadmium accumulation and oxidative stress.
The molecular basis for the high toxicity of cadmium is unclear. Here, Begg et al. use the bacterium Streptococcus pneumoniae as a model system, and show that cadmium uptake increases sensitivity to oxidative stress by reducing intracellular concentrations of manganese and zinc through different mechanisms.
PMCID: PMC4366526  PMID: 25731976
3.  Exploring the correlation between the sequence composition of the nucleotide binding G5 loop of the FeoB GTPase domain (NFeoB) and intrinsic rate of GDP release 
Bioscience Reports  2014;34(6):e00158.
GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role; a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the individual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.
PMCID: PMC4266920  PMID: 25374115
crystal structure; GDP release; GTPase; sequence motif; stopped flow; GPCR, G protein-coupled receptor; EcNFeoB, Escherichia coli FeoB; StNFeoB, Streptococcus thermophiles FeoB; TEV, tobacco etch virus
4.  Structure of an atypical FeoB G-domain reveals a putative domain-swapped dimer 
FeoB is a transmembrane protein involved in ferrous iron uptake in prokaryotic organisms. Here, the structure of the cytoplasmic domain of FeoB from Gallionella capsiferriformans is reported.
FeoB is a transmembrane protein involved in ferrous iron uptake in prokaryotic organisms. FeoB comprises a cytoplasmic soluble domain termed NFeoB and a C-terminal polytopic transmembrane domain. Recent structures of NFeoB have revealed two structural subdomains: a canonical GTPase domain and a five-helix helical domain. The GTPase domain hydrolyses GTP to GDP through a well characterized mechanism, a process which is required for Fe2+ transport. In contrast, the precise role of the helical domain has not yet been fully determined. Here, the structure of the cytoplasmic domain of FeoB from Gallionella capsiferriformans is reported. Unlike recent structures of NFeoB, the G. capsiferriformans NFeoB structure is highly unusual in that it does not contain a helical domain. The crystal structures of both apo and GDP-bound protein forms a domain-swapped dimer.
PMCID: PMC3614164  PMID: 23545645
FeoB; domain-swapped dimer; NFeoB; Gallionella capsiferriformans
5.  The structure of an N11A mutant of the G-protein domain of FeoB 
The structure of NFeoB from S. thermophilus harbouring an N11A mutation has been determined to 1.85 Å resolution.
The uptake of ferrous iron in prokaryotes is mediated by the G-protein-coupled membrane protein FeoB. The protein contains two N-terminal soluble domains that are together called ‘NFeoB’. One of these is a G-protein domain, and GTP hydrolysis by this domain is essential for iron transport. The GTPase activity of NFeoB is accelerated in the presence of potassium ions, which bind at a site adjacent to the nucleotide. One of the ligands at the potassium-binding site is a conserved asparagine residue, which corresponds to Asn11 in Streptococcus thermophilus NFeoB. The structure of an N11A S. thermophilus NFeoB mutant has been determined and refined to a resolution of 1.85 Å; the crystals contained a mixture of mant-GDP-bound and mant-GMP-bound protein. The structure demonstrates how the use of a derivatized nucleotide in cocrystallization experiments can facilitate the growth of diffraction-quality crystals.
PMCID: PMC3232127  PMID: 22139154
FeoB; G-protein domain; Streptococcus thermophilus
6.  The Initiation of GTP Hydrolysis by the G-Domain of FeoB: Insights from a Transition-State Complex Structure 
PLoS ONE  2011;6(8):e23355.
The polytopic membrane protein FeoB is a ferrous iron transporter in prokaryotes. The protein contains a potassium-activated GTPase domain that is essential in regulating the import of iron and conferring virulence to many disease-causing bacteria. However, the mechanism by which the G-domain of FeoB hydrolyzes GTP is not well understood. In particular, it is not yet known how the pivotal step in GTP hydrolysis is achieved: alignment of a catalytic water molecule. In the current study, the crystal structure of the soluble domains from Streptococcus thermophilus FeoB (NFeoBSt) in complex with the activating potassium ion and a transition-state analogue, GDP⋅AlF4−, reveals a novel mode of water alignment involving contacts with the protein backbone only. In parallel to the structural studies, a series of seven mutant proteins were constructed that targeted conserved residues at the active site of NFeoBSt, and the nucleotide binding and hydrolysis properties of these were measured and compared to the wild-type protein. The results show that mutations in Thr35 abolish GTPase activity of the protein, while other conserved residues (Tyr58, Ser64, Glu66 and Glu67) are not required for water alignment by NFeoBSt. Together with the crystal structure, the findings suggest a new mechanism for hydrolysis initiation in small G-proteins, in which the attacking water molecule is aligned by contacts with the protein backbone only.
PMCID: PMC3153494  PMID: 21858085
7.  Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus 
The EMBO Journal  2009;28(23):3771-3779.
Vacuolar-type ATPases (V-ATPases) exist in various cellular membranes of many organisms to regulate physiological processes by controlling the acidic environment. Here, we have determined the crystal structure of the A3B3 subcomplex of V-ATPase at 2.8 Å resolution. The overall construction of the A3B3 subcomplex is significantly different from that of the α3β3 sub-domain in FoF1-ATP synthase, because of the presence of a protruding ‘bulge' domain feature in the catalytic A subunits. The A3B3 subcomplex structure provides the first molecular insight at the catalytic and non-catalytic interfaces, which was not possible in the structures of the separate subunits alone. Specifically, in the non-catalytic interface, the B subunit seems to be incapable of binding ATP, which is a marked difference from the situation indicated by the structure of the FoF1-ATP synthase. In the catalytic interface, our mutational analysis, on the basis of the A3B3 structure, has highlighted the presence of a cluster composed of key hydrophobic residues, which are essential for ATP hydrolysis by V-ATPases.
PMCID: PMC2775895  PMID: 19893485
crystal structure; FoF1; proton pump; rotary motor; V-ATPase
8.  Thioredoxin A Active-Site Mutants Form Mixed Disulfide Dimers That Resemble Enzyme–Substrate Reaction Intermediates 
Journal of Molecular Biology  2008;379(3-2):520-534.
Thioredoxin functions in nearly all organisms as the major thiol–disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme–substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.
PMCID: PMC2896474  PMID: 18455736
disulfide; thioredoxin; Bacillus; structure; dimer; TrxA, thioredoxin A; BsTrxA, Bacillus subtilis TrxA; ArsC, arsenate reductase; BASI, barley α-amylase/subtilisin inhibitor; PDB, Protein Data Bank; AMS, 4-acetamido-4′-maleimidyl-stilbene-2,2′-disulfonate; NF-κB, nuclear factor κB; ESRF, European Synchrotron Radiation Facility
9.  Structure of the T109S mutant of Escherichia coli dihydroorotase complexed with the inhibitor 5-­fluoroorotate: catalytic activity is reflected by the crystal form 
A single-point mutant (T109S) of E. coli dihydroorotase initially crystallizes so that the two monomers of the dimer are related by a crystallographic twofold axis. In the presence of substrate, conversion to the previously observed asymmetric dimer with substrate bound in one subunit and product in the other is observed.
Crystals of a single-point mutant (T109S) of Escherichia coli dihydroorotase (DHOase) with diminished activity grown in the presence of l-dihydroorotate (l-DHO) are tetragonal, with a monomer in the asymmetric unit. These crystals are extremely unstable and disintegrate shortly after formation, which is followed by the growth of orthorhombic crystals from the remnants of the tetragonal crystals or at new nucleation sites. Orthorhombic crystals, for which a structure has previously been reported [Thoden et al. (2001 ▶), Biochemistry, 40, 6989–6997; Lee et al. (2005 ▶), J. Mol. Biol. 348, 523–533], contain a dimer of DHOase in the asymmetric unit; the active site of one monomer contains the substrate N-carbamyl-l-aspartate (l-CA-asp) and the active site of the other monomer contains the product of the reaction, l-DHO. In the subunit with l-­DHO in the active site, a surface loop (residues 105–115) is ‘open’. In the other subunit, with l-CA-asp in the active site, the loop folds inwards, forming specific hydrogen bonds from the loop to the l-CA-asp. The tetragonal crystal form can be stabilized by crystallization in the presence of the inhibitor 5-fluoroorotate (FOA), a product (l-DHO) mimic. Crystals of the complex of T109S DHOase with FOA are tetragonal, space group P41212, with unit-cell parameters a = b = 72.6, c = 176.1 Å. The structure has been refined to R and R free values of 0.218 and 0.257, despite severe anisotropy of the diffraction. In this structure, the flexible loops are both in the ‘open’ conformation, which is consistent with FOA, like l-­DHO, binding at both sites. The behaviour of the T109S mutant crystals of DHOase in the presence of l-DHO is explained by initial binding of l-DHO to both subunits, followed by slow conversion to l-CA-asp, with consequent movement of the flexible loop and dissolution of the crystals. Orthorhombic crystals are then able to grow in the presence of l-DHO and l-CA-asp.
PMCID: PMC2330171  PMID: 17329804
dihydroorotase; conformational change; loop movement; catalytic state; crystal contacts; crystal instability

Results 1-9 (9)