Backbone assignments, and effect of Asn deamidation, of the N-terminal region of the partitioning protein IncC1 from the plasmid RK2.

IncC from the low-copy number plasmid RK2, is a member of the ParA family of proteins required for partitioning DNA in many bacteria and plasmids. It is an ATPase that binds DNA and its ParB protein partner, KorB. Together, the proteins move replicated DNA to appropriate cellular positions, so that each daughter cell inherits a copy on cell division. IncC from RK2 is expressed in two forms. IncC2 is homologous to bacterial ParA proteins, while IncC1 has an N-terminal extension of 105 amino acids and is similar in length to ParA homologues in other plasmids. We have been examining the role of this extension, here called IncC NTD. We present its backbone NMR chemical shift assignments and show that it is entirely intrinsically disordered. The assignments were achieved using C-detected, CON-based spectra, complemented by HNN spectra to obtain connectivities from three adjacent amino acids. We also observed evidence of deamidation of the protein at a GNGG sequence, to give isoAsp, giving 2 sets of peaks for residues up to 5 amino acids on either side of the modification. We have assigned resonances from around the position of modification for this form of the protein.

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

Direct positive selection for improved nitroreductase variants using SOS triggering of bacteriophage lambda lytic cycle.

Expression of prodrug-activating enzymes that convert non-toxic substrates to cytotoxic derivatives is a promising strategy for cancer gene therapy. However, their catalytic activity with unnatural, prodrug substrates is often suboptimal. Efforts to improve these enzymes have been limited by the inability to select directly for increased prodrug activation. We have focussed on developing variants of Escherichia coli (E. coli) nitroreductase (NTR) with improved ability to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), and describe here a novel, direct, positive selection for improved enzymes that exploits the alternative life cycles of bacteriophage lambda. In lambda lysogens of E. coli, the activation of the prodrug CB1954 by NTR triggers the SOS response to DNA damage, switching integrated lambda prophages into lytic cycle. This provides a direct, positive selection for phages encoding improved NTR variants, as, upon limiting exposure of lysogenized E. coli to CB1954, only those encoding the most active enzyme variants are triggered into lytic cycle, allowing their selective recovery. We exemplify the selection by isolating highly improved 'turbo-NTR' variants from a library of 6.8 x 10(5) clones, conferring up to 50-fold greater sensitivity to CB1954 than the wild type. Carcinoma cells infected with adenovirus expressing T41Q/N71S/F124T-NTR were sensitized to CB1954 concentrations 40- to 80-fold lower than required with WT-NTR.

A simple mechanism for co-dependence on two activators at an Escherichia coli promoter.

The Escherichia coli melAB promoter is co-dependent upon two transcription activators, MelR and the cyclic AMP receptor protein, CRP. In this study we demonstrate positive co-operativity between the binding of MelR and CRP at the melAB promoter, which provides a simple mechanism for its co-dependence. MelR binds to four sites, centred at positions -42.5, -62.5, -100.5 and -120.5 relative to the melAB transcription start point. When MelR is pre-bound, CRP is able to bind to a target located between MelR at positions -62.5 and -100.5. This increases the occupation of the two downstream sites for MelR, which is essential for transcription activation. We have identified residues within activating region 1 (AR1) of CRP that are important in transcription activation of the melAB promoter. At simple CRP-dependent promoters, the surface of CRP containing these residues is involved in contacting the RNA polymerase alpha subunit. Our results show that, at the melAB promoter, the surface of CRP containing AR1 contacts MelR rather than RNA polymerase. Thus, MelR and CRP activate transcription by a novel mechanism in which they bind co-operatively to adjacent sites and form a bacterial enhanceosome.

The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution.

Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 A, 1.8 A and 2.4 A, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta-sheet surrounded by alpha-helices. Helices F and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.

Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein.

MelR is a melibiose-triggered transcription activator that belongs to the AraC family of transcription factors. Using purified Escherichia coli RNA polymerase and a cloned DNA fragment carrying the entire melibiose operon intergenic region, we have demonstrated in vitro open complex formation and activation of transcription initiation at the melAB promoter. This activation is dependent on MelR and melibiose. These studies also show that the cyclic AMP receptor protein (CRP) interacts with the melAB promoter and increases MelR-dependent transcription activation. DNAase I footprinting has been exploited to investigate the location of MelR-and CRP-binding sites at the melAB promoter. We showed previously that MelR binds to two identical 18 bp target sequences centred at position -100.5 (Site 1) and position -62.5 (Site 2). In this work, we show that MelR additionally binds to two other related 18 bp sequences: Site 1', centred at position -120.5, located immediately upstream of Site 1, and Site R, at position -238.5, which overlaps the transcription start site of the divergent melR promoter. MelR can bind to Site 1', Site 1, Site 2 and Site R, in both the absence and the presence of melibiose. However, in the presence of melibiose, MelR also binds to a fifth site (Site 2', centred at position -42.5) located immediately downstream of Site 2, and overlapping the -35 region of the melAB promoter. Additionally, although CRP is unable to bind to the melAB promoter in the absence of MelR, in the presence of MelR, it binds to a site located between MelR binding Site 1 and Site 2. Thus, tandem-bound MelR recruits CRP to the MelR. We propose that expression from the melAB promoter has an absolute requirement for MelR binding to Site 2'. Optimal expression of the melAB promoter requires Sites 1', Site 1, Site 2 and Site 2'; CRP acts as a 'bridge' between MelR bound at Sites 1' and 1 and at Sites 2 and 2', increasing expression from the melAB promoter. In support of this model, we show that improvement of the base sequence of Site 2' removes the requirement for Site 1' and Site 1, and short circuits the effects of CRP.

Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop.

The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and -10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and -10 region. Parallels with the Escherichia coli araC-araBAD regulatory region are discussed.

Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences.

The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity. Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial. In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants. Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies. Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and aroL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super-repressor Trp mutants. This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes. In contrast, DNA containing containing a symmetrized trpR operator sequence, trpRs, gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer-DNA complex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed. This shows that the binding of the second dimer is sequence selective. We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences. This allows it to co-ordinately control promoters of different intrinsic strength. This mechanism may be of importance in a number of promoters that bind multiple effector molecules.

Secondary structure of the C-terminal DNA-binding domain of the transcriptional activator NifA from Klebsiella pneumoniae: spectroscopic analyses.

The conformation of the C-terminal DNA-binding domain of the transcriptional activator NifA from Klebsiella pneumoniae has been probed by circular dichroism (CD), Fourier-transformed infrared (FT-IR), and nuclear magnetic resonance (NMR) spectroscopy in combination. Secondary structure prediction suggests that the C-terminal half of the domain contains three alpha-helices. The spectra show that the domain is folded in the absence of DNA and of the N-terminal and central domains of NifA. The three spectroscopic techniques suggest slightly different proportions of secondary structural elements but all suggest that it contains about 33% alpha-helix. These results are in agreement with a previous prediction suggesting that NifA contains a helix-turn-helix motif and with the amount of alpha-helix predicted. The environment of the aromatic residues was examined by CD and NMR spectroscopy, which suggest that one or both of the tryptophan residues are involved in the tertiary structure of the protein but that the tyrosine residue in the helix-turn-helix motif is solvent exposed and so available to bind to DNA. The thermal melting profiles and pH-dependent structural changes were also examined by CD spectroscopy. This technique indicates that at low pH there is an increase in the secondary structure and interactions contributing to the tertiary structure. Many of the acidic residues are predicted to be on a single helix, before the helix-turn-helix motif, which may therefore be important for maintaining the structure and function of the C-terminal peptide; alternatively, the N-terminal half of the domain may become more folded at low pH.

DNA binding and DNA bending by the MelR transcription activator protein from Escherichia coli.

The Escherichia coli melR gene encodes MelR protein which is a member of the AraC/XylS family of bacterial transcription activators. The function of MelR was investigated by making a targeted deletion in the melR gene of the Escherichia coli chromosome. MelR is a transcription activator essential for melibiose- dependent expression of the melAB operon which is needed for bacterial growth with melibiose as a carbon source. To investigate the interactions of MelR at the melAB promoter, both full length MelR and a shortened derivative, MelR173, containing the C-terminal DNA-binding domain, were purified as fusions to glutathione- S -transferase. Circular permutation studies show that both full-length MelR and MelR173 induce an apparent bend upon binding to target sites at the melAB promoter. Bound full-length MelR, but not MelR173, can oligomerise to form larger complexes that are likely to be involved in transcription activation.

NMR studies of the Escherichia coli Trp repressor.trpRs operator complex.

To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H-NMR spectroscopy. We report here the 1H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.

Three-dimensional structure of the RGD-containing snake toxin albolabrin in solution, based on 1H NMR spectroscopy and simulated annealing calculations.

Albolabrin is a snake toxin that contains a RGD-(Arg-Gly-Asp) sequence motif and competes with fibrinogen to bind to the integrin alpha IIb beta 3 (GpIIb-IIIa) on platelets. It thus inhibits platelet aggregation and cell-cell adhesion. It shows a high sequence similarity to other disintegrins, yet the reported disulfide bonding pattern for this peptide differs from that of others in this family. Recently we reported the assignment of the 1H-NMR spectrum of albolabrin and a preliminary description of its secondary structure [Jaseja, M., Smith, K.J., Lu, X. Williams, J.A., Trayer, H., Trayer, I.P. & Hyde, E.I. (1993) Eur. J. Biochem. 218, 853-860]. Here we present a more detailed description of the secondary and the tertiary structure, based on the 1H NMR results and simulated annealing methods. The structure of albolabrin in solution was calculated using 318 distance and 18 dihedral angle restraints. The average atomic RMS deviation between 12 refined structures and the mean structure was 3.1 A for the backbone. The protein appears to be highly mobile. Its structure is dominated by a series of turns and by three hairpins, each with a short region of distorted antiparallel beta-pleated sheet, held together by six disulfide bridges. The most well defined area is the hydrophobic core, residues 21-47 and 57-67, which is clustered around F40 and has a backbone atomic RMS deviation of only 1.3 A from the mean structure. The RGD adhesion sequence is found at the highly mobile tip of one of the beta-hairpins, protruding from the body of the protein. Many of these structural features are similar to those of other disintegrins, and differences in the disulfide bonding pattern of the disintegrins can be accomodated without significant energy penalty. Comparison of this structure with other proteins of similar function suggests that it is the RGD-loop, rather than the precise technology of the proteins, that is important to antagonist activity.

The Escherichia coli MelR transcription activator: production of a stable fragment containing the DNA-binding domain.

A set of nested deletions has been made in the Escherichia coli melR gene, encoding the MelR transcription activator protein. Expression of the resulting melR derivatives led to the production of nine MelR proteins with N-terminal deletions of different lengths. The properties of the shortened proteins have been studied both in vivo and in vitro. None of the truncated proteins activate transcription from the E.coli melAB promoter but three; MelR220, MelR183 and MelR173, inhibit activation of the melAB promoter by chromosomally-encoded full-length MelR. In gel retardation assays, both MelR183 and MelR173 clearly retard DNA fragments carrying the melAB promoter. MelR173 has been overproduced in a T7 expression system and shown to be stable in vivo for up to 2 h. DNAase I footprinting assays of partially purified protein show that it binds to the melAB promoter, protecting the same sites as the full-length protein. This fragment may be suitable for further structure/function studies of this class of transcription activator.

1H-NMR assignments and secondary structure of dendroaspin, an RGD-containing glycoprotein IIb-IIIa (alpha IIb-beta 3) antagonist with a neurotoxin fold.

Dendroaspin, also referred to as mambin, was originally isolated from the venom of the Elapidae snake Dendroaspis jamesoni kaimose. It shares a high level of sequence similarity with the short-chain neurotoxins found in other Elapidae but displays approximately 1000-fold lower neurotoxin activity than the closely related protein erabutoxin b. However, unlike neurotoxins, it contains an RGD (Arg-Gly-Asp) motif and functions as an antagonist of platelet aggregation and cell-cell adhesion of comparable potency to the disintegrins from the venoms of Viperidae. We have determined the secondary structure of dendroaspin using 1H-NMR spectroscopy. Its structure resembles that of the short-chain neurotoxins, with three loops extending from a disulphide-bridged core; however, the strands of the triple-stranded beta-sheet are shorter and the loop containing the RGD sequence is moved away from this sheet. The structure bears little resemblance to that of the disintegrins, except in the RGD-containing loop, suggesting that this loop may be of prime importance in its inhibitory function. Comparison of this preliminary structure with that of the neurotoxins and disintegrins furthers our understanding of the mechanism of integrin antagonists and shows how the neurotoxin fold can be manipulated to give a variety of inhibitors.

Three-dimensional structure of the RGD-containing neurotoxin homologue dendroaspin.

Dendroaspin is a short chain neurotoxin homologue from the venom of Elapidae snakes, which lacks neurotoxicity. Unlike neurotoxins, it contains an Arg-Gly-Asp-(RGD)-motif and functions as an inhibitor of platelet aggregation and platelet adhesion with comparable potency to the disintegrins from the venoms of Viperidae. We have determined the structure of dendroaspin in solution using NMR spectroscopy. The structure contains a core similar to that of short chain neurotoxins, but with a novel arrangement of loops and a solvent-exposed RGD-motif. Dendroaspin is thus an integrin antagonist with a well defined fold different from that of the disintegrins, based on the neurotoxin scaffold.

1H-NMR studies and secondary structure of the RGD-containing snake toxin, albolabrin.

Albolabrin is a naturally occurring peptide from snake venom containing the sequence Arg-Gly-Asp (RGD). It inhibits platelet aggregation by blocking the binding of fibrinogen to the glycoprotein Gp IIb-IIIa, on the surface of activated platelets. Albolabrin consists of 73 residues with six intramolecular disulphide bonds. The 1H-NMR spectrum of albolabrin has been assigned using homonuclear two-dimensional techniques and its secondary structure determined. Like kistrin and echistatin, two related peptides from snake venom, albolabrin appears to have little regular secondary structure in solution. Several bends and two short distorted beta sheets are observed. The RGD sequence, important for binding to the receptor, lies in a mobile loop joining two strands of one of these beta sheets. This loop undergoes a pH-dependent conformational change.

NMR studies of the activation of the Escherichia coli trp repressor.

The Escherichia coli trp repressor binds to the trp operator in the presence of tryptophan, thereby inhibiting tryptophan biosynthesis. Tryptophan analogues lacking the alpha-amino group act as inducers of trp operon expression. We have used one- and two-dimensional 1H-NMR spectroscopy to compare the binding to the repressor of the corepressors L-tryptophan, D-tryptophan and 5-methyl-DL-tryptophan with that of the inducer indole-3-propionic acid. We have determined the chemical shifts of the indole ring protons of the ligands when bound to the protein, principally by magnetization-transfer experiments. The chemical shifts of the indole NH and C4 protons differ between corepressors and inducer. At the same time, the pattern of intermolecular NOE between protons of the protein and those of the ligand also differ between the two classes of ligand. These two lines of evidence indicate that corepressors and inducers bind differently in the binding site, and the evidence suggests that the orientation of the indole ring in the binding site differs by approximately 180 degrees between the two kinds of ligand. This is in contrast to a previous solution study [Lane, A.N. (1986) Eur. J. Biochem. 157, 405-413], but consistent with recent X-ray crystallographic work [Lawson, C.L. & Sigler, P.B. (1988) Nature 333, 869-871]. D-Tryptophan and 5-methyltryptophan, which are more effective corepressors than L-tryptophan, bind similarly to L-tryptophan. The indole ring of D-tryptophan appears to bind in essentially the same orientation as that of the L isomer. There are, however, some differences in chemical shifts and NOE for 5-methyltryptophan, which indicate that there are significant differences between the two corepressors L-tryptophan and 5-methyltryptophan in the orientation of the indole ring within the binding site.

1H NMR studies of the glucocorticoid receptor DNA-binding domain: sequential assignments and identification of secondary structure elements.

Two protein fragments containing the DNA-binding domain (DBD) of the glucocorticoid receptor (GR) have been studied by two-dimensional 1H NMR spectroscopy. The two peptides (93 and 115 residues, respectively) contain a common segment corresponding to residues C440-I519 of the rat GR or residues C421-I500 of the human GR and include two Zn-binding "finger" domains. The structures of this segment are almost identical in the two protein fragments, as judged from chemical shifts and sequential NOE connectivities. More than 90% of all observable 1H resonances within a 71-residue segment encompassing C440-R510 (rat GR) could be sequentially assigned by standard techniques, and stereospecific assignments could be made for the methyl groups in four valine residues within this segment. Sequential NOE connectivities indicate several elements of secondary structure including two alpha-helical segments consisting of residues S459-E469 and P493-G504, a type I reverse turn between residues R479 and C482, a type II reverse turn between residues L475 and G478, and several regions of extended peptide conformation. No evidence for alpha-helical conformation was found within the two putative zinc-finger domains, indicating that the structures of these domains differ from that of TFIIIA-type zinc fingers. The observation of some very slowly exchanging amide protons in the N-terminal (CI) domain of the DBD in combination with slow rotation of the Y452 aromatic ring indicates that this domain has a restricted conformational flexibility compared to the C-terminal (CII) domain. We also observe several long-range NOE connectivities within C440-R510, suggesting that the sequential assignments presented here will provide a basis for a complete structure determination of this segment of the GR.

NMR studies of the Escherichia coli trp aporepressor. Sequence-specific assignment of the aromatic proton resonances.

The resonances in the aromatic region of the 1H-NMR spectrum of the Escherichia coli trp aporepressor have been assigned to amino acid type by two-dimensional correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA) spectroscopy and nuclear Overhauser enhancement spectroscopy (NOESY) techniques and studies of the pH dependence of the chemical shifts, in combination with selective deuteration of the protein. Complete sequence-specific assignments of the aromatic resonances have been made by comparing the observed inter-residue NOEs with those expected on the basis of the crystal structure of the protein [Zhang, R.-G., Joachimiak, A., Lawson, C.L., Shevitz, R.W., Otwinowski, Z. & Sigler, P.B. (1987) Nature 327, 591-597]. The latter experiments have also permitted the sequence-specific assignment of some of the high-field methyl resonances. The complete assignment of the aromatic region of the spectrum, in particular of resonances from residues at the dimer interface, opens the way to detailed studies of the conformational effects of corepressor and operator binding.