Oxygen-insensitive nitroreductase E. coli NfsA, but not NfsB, is inhibited by fumarate.

Escherichia coli NfsA and NfsB are founding members of two flavoprotein families that catalyze the oxygen-insensitive reduction of nitroaromatics and quinones by NAD(P)H. This reduction is required for the activity of nitrofuran antibiotics and the enzymes have also been proposed for use with nitroaromatic prodrugs in cancer gene therapy and biocatalysis, but the roles of the proteins in vivo in bacteria are not known. NfsA is NADPH-specific whereas NfsB can also use NADH. The crystal structures of E. coli NfsA and NfsB and several analogs have been determined previously. In our crystal trials, we unexpectedly observed NfsA bound to fumarate. We here present the X-ray structure of the E. coli NfsA-fumarate complex and show that fumarate acts as a weak inhibitor of NfsA but not of NfsB. The structural basis of this differential inhibition is conserved in the two protein families and occurs at fumarate concentrations found in vivo, so impacting the efficacy of these proteins.

Structure and Dynamics of Three Escherichia coli NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954.

Escherichia coli NfsB has been studied extensively for its potential for cancer gene therapy by reducing the prodrug CB1954 to a cytotoxic derivative. We have previously made several mutants with enhanced activity for the prodrug and characterised their activity in vitro and in vivo. Here, we determine the X-ray structure of our most active triple and double mutants to date, T41Q/N71S/F124T and T41L/N71S. The two mutant proteins have lower redox potentials than wild-type NfsB, and the mutations have lowered activity with NADH so that, in contrast to the wild-type enzyme, the reduction of the enzyme by NADH, rather than the reaction with CB1954, has a slower maximum rate. The structure of the triple mutant shows the interaction between Q41 and T124, explaining the synergy between these two mutations. Based on these structures, we selected mutants with even higher activity. The most active one contains T41Q/N71S/F124T/M127V, in which the additional M127V mutation enlarges a small channel to the active site. Molecular dynamics simulations show that the mutations or reduction of the FMN cofactors of the protein has little effect on its dynamics and that the largest backbone fluctuations occur at residues that flank the active site, contributing towards its broad substrate range.

The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN.

NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH- to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.

Structure of SPH (self-incompatibility protein homologue) proteins: a widespread family of small, highly stable, secreted proteins.

SPH (self-incompatibility protein homologue) proteins are a large family of small, disulfide-bonded, secreted proteins, initially found in the self-incompatibility response in the field poppy (Papaver rhoeas), but now known to be widely distributed in plants, many containing multiple members of this protein family. Using the Origami strain of Escherichia coli, we expressed one member of this family, SPH15 from Arabidopsis thaliana, as a folded thioredoxin fusion protein and purified it from the cytosol. The fusion protein was cleaved and characterised by analytical ultracentrifugation, circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. This showed that SPH15 is monomeric and temperature stable, with a ß-sandwich structure. The four strands in each sheet have the same topology as the unrelated proteins: human transthyretin, bacterial TssJ and pneumolysin, with no discernible sequence similarity. The NMR-derived structure was compared with a de novo model, made using a new deep learning algorithm based on co-evolution/correlated mutations, DeepCDPred, validating the method. The DeepCDPred de novo method and homology modelling to SPH15 were then both used to derive models of the 3D structure of the three known PrsS proteins from P. rhoeas, which have only 15-18% sequence homology to SPH15. The DeepCDPred method gave models with lower discreet optimised protein energy scores than the homology models. Three loops at one end of the poppy structures are postulated to interact with their respective pollen receptors to instigate programmed cell death in pollen tubes.

Rationalisation of a mechanism for sensing single point variants in target DNA using anthracene-tagged base discriminating probes.

The labelling of DNA oligonucleotides with signalling groups that give a unique response to duplex formation depending on the target sequence is a highly effective strategy in the design of DNA-based hybridisation sensors. A key challenge in the design of these so-called base discriminating probes (BDPs) is to understand how the local environment of the signalling group affects the sensing response. The work herein describes a comprehensive study involving a variety of photophysical techniques, NMR studies and molecular dynamics simulations, on anthracene-tagged oligonucleotide probes that can sense single base changes (point variants) in target DNA strands. A detailed analysis of the fluorescence sensing mechanism is provided, with a particular focus on rationalising the high dependence of this process on not only the linker stereochemistry but also the site of nucleobase variation within the target strand. The work highlights the various factors and techniques used to respectively underpin and rationalise the BDP approach to point variant sensing, which relies on different responses to duplex formation rather than different duplex binding strengths.

Intrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA.

The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.

Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator.

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.

The 3D-structure, kinetics and dynamics of the E. coli nitroreductase NfsA with NADP+ provide glimpses of its catalytic mechanism.

Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics, we show that the Escherichia coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP+ shows that a mobile loop forms a phosphate-binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP+ is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half-of-sites mechanism.

Order and disorder in the domain organization of the plasmid partition protein KorB.

The plasmid partition protein KorB has a dual role: it is essential for the correct segregation of the low copy number broad host range RK2 plasmid while also being an important regulator of transcription. KorB belongs to the ParB family of proteins, and partitioning in RK2 has been studied as a simplified model of bacterial chromosome segregation. Structural information on full-length ParB proteins is limited, mainly due to the inability to grow crystals suitable for diffraction studies. We show, using CD and NMR, that KorB has regions of significant intrinsic disorder and hence it adopts a multiplicity of conformations in solution. The biophysical data are consistent with bioinformatic predictions based on the amino acid sequence that the N-terminal region and also the region between the central DNA-binding domain and the C-terminal dimerization domain are intrinsically disordered. We have used small angle x-ray scattering data to determine the ensemble of solution conformations for KorB and selected deletion mutants, based on models of the known domain structures. This conformational range of KorB is likely to be biologically required for DNA partitioning and for binding to a diverse set of partner proteins.

Backbone assignment of E. coli NfsB and the effects of addition of the cofactor analogue nicotinic acid.

E. coli nitroreductase NfsB (also called NfnB) has been studied extensively, largely due to its potential for cancer gene therapy. A homodimeric flavoprotein of 216 residues, it catalyses the reduction of nitroaromatics to cytotoxic hydroxylamines by NADH and NADPH and also the reduction of quinones to hydroxyquinones. Its role in vivo is not known but it is postulated to be involved in reducing oxidative stress. The crystal structures of the wild type protein and several homologues have been determined in the absence and presence of ligands, including nicotinate as a mimic of the headpiece of the nicotinamide cofactors. There is little effect on the overall structure of the protein on binding ligands, but, from the B factors, there appears to be a decrease in mobility of 2 helices near the active site. As a first step towards examining the dynamics of the protein in solution with and without ligand, we have assigned the backbone 13C, 15N, and 1HN resonances of NfsB and examined the effect of the binding of nicotinate on the amide 15N, and 1HN shifts.

Steady-state and stopped-flow kinetic studies of three Escherichia coli NfsB mutants with enhanced activity for the prodrug CB1954.

The enzyme nitroreductase, NfsB, from Escherichia coli has entered clinical trials for cancer gene therapy with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, CB1954 is a poor substrate for the enzyme. Previously we made several NfsB mutants that show better activity with CB1954 in a cell-killing assay in E. coli. Here we compare the kinetic parameters of wild-type NfsB with CB1954 to those of the most active single, double, and triple mutants isolated to date. For wild-type NfsB the global kinetic parameters for both k(cat) and K(m) for CB1954 are about 20-fold higher than previously estimated; however, the measured specificity constant, k(cat)/K(m) is the same. All of the mutants are more active with CB1954 than the wild-type enzyme, the most active mutant showing about 100-fold improved specificity constant with CB1954 over the wild-type protein with little effect on k(cat). This enhancement in specificity constants for the mutants is not seen with the antibiotic nitrofurazone as substrate, leading to reversed nitroaromatic substrate selectivity for the double and triple mutants. However, similar enhancements in specificity constants are found with the quinone menadione. Stopped-flow kinetic studies suggest that the rate-determining step of the reaction is likely to be the release of products. The most active mutant is also selective for the 4-nitro group of CB1954, rather than the 2-nitro group, giving the more cytotoxic reduction product. The double and triple mutants should be much more effective enzymes for use with CB1954 in prodrug-activation gene therapy.

A single aromatic residue in transcriptional repressor protein KorA is critical for cooperativity with its co-regulator KorB.

A central feature of broad host range IncP-1 plasmids is the set of regulatory circuits that tightly control plasmid core functions under steady-state conditions. Cooperativity between KorB and either KorA or TrbA repressor proteins is a key element of these circuits and deletion analysis has implicated the conserved C-terminal domain of KorA and TrbA in this interaction. By NMR we show that KorA and KorB interact directly and identify KorA amino acids that are affected on KorB binding. Studies on mutants showed that tyrosine 84 (or phenylalanine, in some alleles) is dispensable for repressor activity but critical for the specific interaction with KorB in both in vivo reporter gene assays and in vitro electrophoretic mobility shift and co-purification assays. This confirms that direct and specific protein-protein interactions are responsible for the cooperativity observed between KorB and its corepressors and lays the basis for determining the biological importance of this cooperativity.

1H, 13C and 15N NMR assignments of self-incompatibility protein homologue 15 from Arabidopsis thaliana.

The SPH proteins are a large family of small, disulphide-bonded, secreted proteins, originally found to be involved in the self-incompatibility response in the field poppy (Papaver rhoeas). They are now known to be widely distributed in plants, many containing multiple members of this protein family. Apart from the PrsS proteins in Papaver the function of these proteins is unknown but they are thought to be involved in plant development and cell signalling. There has been no structural study of SPH proteins to date. Using the Origami strain of E. coli, we cloned and expressed one member of this family, SPH15 from Arabidopsis thaliana, as a folded thioredoxin-fusion protein, purified it from the cytosol, and cleaved it to obtain the secreted protein. We here report the assignment of the NMR spectra of SPH15, which contains 112 residues plus three N-terminal amino acids from the vector. The secondary structure propensity from TALOS+ shows that it contains eight beta strands and connecting loops. This is largely in agreement with predictions from the amino acid sequence, which show an additional C-terminal strand.

Kinetic and structural characterisation of Escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate CB1954.

Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme-prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.

1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, beta-arrestin and RACK1.

The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, betaarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the beta-arrestin binding site. (1)H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic alpha-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix-helix interaction shown for G(gamma) binding to the WD-repeat protein, G(beta). A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in beta-arrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with beta-arrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to beta-arrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both beta-arrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the beta(2)-adrenergic receptors (beta(2)AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards.

Secondary structure and DNA binding by the C-terminal domain of the transcriptional activator NifA from Klebsiella pneumoniae.

The NifA protein of Klebsiella pneumoniae is required for transcriptional activation of all nitrogen fixation (nif) operons except the regulatory nifLA genes. At these operons, NifA binds to an upstream activator sequence (UAS), with the consensus TGT-N(10)-ACA, via a C-terminal DNA-binding domain (CTD). Binding of the activator to this upstream enhancer-like sequence allows NifA to interact with RNA polymerase containing the alternative sigma factor, sigma(54). The isolated NifA CTD is monomeric and binds specifically to DNA in vitro as shown by DNase I footprinting. Heteronuclear 3D NMR experiments have been used to assign the signals from the protein backbone. Three alpha-helices have been identified, based on secondary chemical shifts and medium range Halpha(i)-NH(i)( + 1), and NH(i)-NH(i)( + 1) NOEs. On addition of DNA containing a half-site UAS, several changes are observed in the NMR spectra, allowing the identification of residues that are most likely to interact with DNA. These occur in the final two helices of the protein, directly confirming that DNA binding is mediated by a helix-turn-helix motif.

DNA binding of the transcription activator protein MelR from Escherichia coli and its C-terminal domain.

MelR is an Escherichia coli transcription factor belonging to the AraC family. It activates expression of the melAB operon in response to melibiose. Full-length MelR (MelR303) binds to two pairs of sites upstream of the melAB transcription start site, denoted sites 1' and 1 and sites 2 and 2', and to a fifth site, R, which overlaps the divergent melR promoter. The C-terminal domain of MelR (MelR173) does not activate transcription. Here we show that, like MelR303, when MelR173 binds to sites 1 and 2 it recruits CRP to bind between these sites. Hence, the C-terminal domain is involved in heterologous interactions. MelR173 binds to the R site, which has 11 of 18 bp identical to sites 1 and 2 but, surprisingly, does not bind to site 1', which has 12 of 18 bp identical, nor to site 2'. Using electrophoretic mobility shift assays, we show that the binding of MelR303 to sites 1' and 2' is due to cooperative binding with the adjacent site. This homologous cooperativity requires the N-terminal domain of the protein. Activation of the melAB promoter requires MelR to occupy site 2', which overlaps the -35 hexamer. Hence, both domains of MelR are required for transcription activation.

Generation of Escherichia coli nitroreductase mutants conferring improved cell sensitization to the prodrug CB1954.

Escherichia coli nitroreductase (NTR) activates the prodrug CB1954 to a cytotoxic derivative, allowing selective sensitization of NTR-expressing cells or tumors to the prodrug. This is one of several enzyme-prodrug combinations that are under development for cancer gene therapy, and the system has now entered clinical trials. Enhancing the catalytic efficiency of NTR for CB1954 could improve its therapeutic potential. From the crystal structure of an enzyme-ligand complex, we identified nine amino acid residues within the active site that could directly influence prodrug binding and catalysis. Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E. coli to CB1954. Amino acid substitutions at six positions conferred markedly greater sensitivity to CB1954 than did the WT enzyme; the best mutants, at residue F124, resulted in approximately 5-fold improvement. Using an adenovirus vector, we introduced the F124K NTR mutant into human SK-OV-3 ovarian carcinoma cells and showed it to be approximately 5-fold more potent in sensitizing the cells to CB1954 at the clinically relevant prodrug concentration of 1 micro M than was the WT enzyme. Enhanced mutant NTRs such as F124K should improve the efficacy of the NTR/CB1954 combination in cancer gene therapy.

Nitroaryl phosphoramides as novel prodrugs for E. coli nitroreductase activation in enzyme prodrug therapy.

Cyclic and acyclic nitroaryl phosphoramide mustard analogues were activated by E. coli nitroreductase, an enzyme explored in GDEPT. The more active acyclic 4-nitrobenzyl phosphoramide mustard (7) showed 167 500x selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC(50) as low as 0.4 nM. This is about 100x more active and 27x more selective than CB1954 (1). The superior activity was attributed to its better substrate activity (k(cat)/K(m) 19x better than 1) and/or excellent cytotoxicity of phosphoramide mustard released.

Synthesis of α-glucan in mycobacteria involves a hetero-octameric complex of trehalose synthase TreS and Maltokinase Pep2.

Recent evidence established that the cell envelope of Mycobacterium tuberculosis, the bacillus causing tuberculosis (TB), is coated by an α-glucan-containing capsule that has been implicated in persistence in a mouse infection model. As one of three known metabolic routes to α-glucan in mycobacteria, the cytoplasmic GlgE-pathway converts trehalose to α(1 → 4),α(1 → 6)-linked glucan in 4 steps. Whether individual reaction steps, catalyzed by trehalose synthase TreS, maltokinase Pep2, and glycosyltransferases GlgE and GlgB, occur independently or in a coordinated fashion is not known. Here, we report the crystal structure of M. tuberculosis TreS, and show by small-angle X-ray scattering and analytical ultracentrifugation that TreS forms tetramers in solution. Together with Pep2, TreS forms a hetero-octameric complex, and we demonstrate that complex formation markedly accelerates maltokinase activity of Pep2. Thus, complex formation may act as part of a regulatory mechanism of the GlgE pathway, which overall must avoid accumulation of toxic pathway intermediates, such as maltose-1-phosphate, and optimize the use of scarce nutrients.