The interaction of the trp repressor from Escherichia coli with the trp operator

We have examined the interaction of the trp repressor from Escherichia coli with a 20 base-pair synthetic operator. Nonspecific binding was relatively strong (Kd = 2 microM), but only weakly sensitive to the concentration of added salt [d log Kd)/(d log [Na]) = -1). 1H-NMR studies indicate that the structure of the repressor is not greatly altered on forming the complex, and that few if any of the lysine and arginine residues make direct contact with the DNA. However, the mobility of one of the two tyrosine residues is significantly decreased in the complex. The repressor makes close contact with the major grooves of the operator such that the base protons are broadened much more than expected on the basis of increased correlation time. There are large, differential changes in chemical shifts of the imino protons on forming the complex, as well as changes in the rate constants for exchange. The fraying of the ends is greatly diminished, consistent with a target size of about 20 base-pairs. The effects of the repressor on the NMR spectra and relaxation rate constants can be interpreted as a change in the conformation of the operator, possibly a kinking in the centre of the molecule.     

Preliminary identification of the secondary structure of the trp repressor from Escherichia coli

The probable secondary structure content of the trp repressor from Escherichia coli has been inferred from NMR and circular dichroic measurements; the results are compared with those of prediction algorithms. 70% of the amide protons have exchange rate constants orders of magnitude smaller than the intrinsic rate constants, identifying them as participating in hydrogen bonds. The exchange rate constants fall into two distinct classes, one having half-lives of 20 min and the other more than 24 h. The latter class, consisting of 50% of all amide protons, indicates a stable core. The exchange data are consistent with circular dichroism and predictions that suggest that about 55% of the peptides from alpha helix, and 20% form beta sheets and turns. The NMR spectrum further indicates that there is little beta sheet, suggesting that the secondary structure class is alpha.     

Functional inferences from crystals of Escherichia coli trp repressor

We have reproducibly grown crystals of L-tryptophan . trp aporepressor and indole-3-propionate . trp aporepressor complexes from Escherichia coli which are suitable for x-ray diffraction analysis. The active repressor, L-tryptophan . aporepressor, crystallizes in both trigonal (P3(1)21 or P3(2)21) and tetragonal (P4(1)22 or P4(3)22) forms which diffract to at least 2.0 and 2.5 A, respectively. The trigonal form has one-half of the functional dimer/asymmetric unit; therefore, the trp repressor molecule has an axis of 2-fold rotational symmetry corresponding to the lattice dyad. The inactive complex, indole-3-propionate . aporepressor, or "pseudorepressor," forms tetragonal crystals that also diffract to at least 2.5 A and are isomorphous to those of the active repressor. Slight differences between their diffraction patterns indicate modest structural differences between active and inactive complexes that are presumably mediated by the alpha-amino group of L-tryptophan and account for operator-specific binding.     

Interaction of the trp repressor from Escherichia coli with a constitutive trp operator.

The mechanisms of the requirement of glucose for steroidogenesis were investigated by monitoring the uptake of the glucose analogue 2-deoxy-D-glucose by rat testis and tumour Leydig cells. The characteristics of glucose transport in both of these cell types were found to resemble those of the facilitated-diffusion systems for glucose found in most other mammalian cells. The Leydig cells took up 2-deoxy-D-glucose but not L-glucose, and the uptake was inhibited by both cytochalasin B and forskolin. In the presence of luteinizing hormone, the rate of 2-deoxy-D-glucose uptake by both cell types was increased by approx. 50%. In addition to D-glucose, it was shown that the Leydig cells could also utilize 3-hydroxybutyrate or glutamine to maintain steroidogenesis.     

The solution structures of Escherichia coli trp repressor and trp aporepressor at an intermediate resolution

We have determined the solution structures and examined the dynamics of the Escherichia coli trp repressor (a 25-kDa dimer), with and without the co-repressor L-tryptophan, from NMR data. This is the largest protein structure thus far determined by NMR. To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing. Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides. Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization. We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van-der-Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid. An examination of these structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3-10. Sequence-specific 1H-NMR assignments and the secondary structure of the holopressor (L-tryptophan-bound form) have been reported previously [C. H. Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990) Biochemistry 29, 6332-6341]. Those for the trp aporepressor (L-tryptophan-free form), made using the same methods and conditions as described in the cited paper, are reported here. The secondary structure of the aporepressor was calculated from sequential and medium-range NOEs and is the same as reported for the holorepressor except that helix E is shorter. The tertiary solution structures for both forms of the repressor were calculated from long-range NOE data.(ABSTRACT TRUNCATED AT 400 WORDS)     

The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid

The binding of the corepressor, L-tryptophan, and an inducer, indole propanoic acid, to the trp repressor from Escherichia coli was studied by absorbance, fluorescence, circular dichroic and proton NMR spectroscopy. The two ligands bind to the same site on the repressor in the same orientation; they are molecular competitors. The binding site is of relatively low polarity and contains at least one methyl group that lies 0.3 nm over the indole moiety near the C5 proton of the bound ligand, and an aromatic residue, probably tyrosine. The dissociation constant was determined as a function of temperature and pH. At 25 degrees C in 0.1 M phosphate buffer, pH 7.6, the dissociation constant is 18 +/- 2 microM for both ligands. In the same buffer system, the van't Hoff enthalpy for dissociation is 35.5 +/- 1 kJ/mol for tryptophan, and 30.5 +/- 2 kJ/mol for indole propanoic acid. The affinity of the repressor for indole propanoic acid is independent of pH in the range 7 less than 10, but decreases four fold for tryptophan in the same range. The amino group of tryptophan makes a significant contribution to its binding affinity. Difference NMR spectra showed that there are few changes of protein resonances on binding ligands. The NMR signals of the bound resonances were assigned by difference and nuclear Overhauser effect spectroscopy. The properties of the bound resonances are consistent with the ligands being largely immobilised within the binding site. The difference spectra, and the known functional differences of the two ligands, suggest that tryptophan induces a slightly different conformational state in the repressor from that induced by indole propanoic acid. There is no evidence for a global transition. The rate of dissociation of ligands is relatively large, being in the range 400-600 s-1.     

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.     

Identification of surface residues in the trp repressor of Escherichia coli

A subset of the spin systems assigned in the 1H NMR spectrum of the trp repressor in the first paper in this series (our penultimate preceding paper in this journal) can be identified as surface or buried residues on the basis of four independent types of measurement: selective spin-lattice relaxation times; the dependence of line widths on temperature and the concentration of manganous ion; fluorescence quenching; and titration behaviour. Criteria are developed for distinguishing surface and buried residues. The significance for the function of DNA binding proteins is discussed.     

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.     

The influence of tryptophan on mobility of residues in the trp repressor of Escherichia coli

The relative mobility of residues in the trp repressor of Escherichia coli has been examined in the absence and presence of the corepressor L-tryptophan by one- and two-dimensional 1H NMR. A comparison of relative intensities of cross peaks in NOESY and COSY spectra allowed a rigid Tyr and a mobile Tyr residue, three mobile Ser residues and three mobile Lys residues to be detected. The two Tyr residues were assigned by selective nitration with tetranitromethane. The singly nitrated molecule (on Tyr7) binds the trp operator with an affinity close to that of the unmodified repressor. Measurements of the intraring cross-relaxation rate constant as a function of temperature for Tyr7 shows the presence of considerable internal motion on the subnanosecond time scale in the flexible N-terminal arm. The order parameter, S2, characterising the motion is 0.35, which increases to about 0.5 in the presence of Trp. Trp decreases both the amplitude of the motion and the rate of the motion. At least three of the six Ser residues of the trp repressor have greater mobility than expected for a rigid body, and two of the Ser residues are sensitive to the presence of Trp. The more mobile Ser residues are probably those on the N-terminal arm and the C-terminal sequence. These results complement the single-crystal X-ray diffraction studies for which the electron density of the first ten and last three amino acid residues is weak. The solution data are consistent with proposals that the flexible N-terminal arm of the trp repressor makes important contacts with the DNA.     

NMR studies of the trp repressor from Escherichia coli. Characterisation and assignments of residue types

High-resolution proton nuclear magnetic resonance spectra of the trp repressor of Escherichia coli under various conditions are reported and analysed. The spectrum of the denatured state agrees with that predicted from the amino acid composition, with the exception of the two histidine residues, which have different chemical shifts although they titrate normally. The spectrum of the native protein shows the presence of extensive secondary and tertiary structure. Using information from chemical shifts, numbers of protons, titration behaviour, homonuclear chemical-shift-correlated spectroscopy and nuclear Overhauser enhancement correlated spectroscopy, most of the aromatic protons have been assigned to residue type. Further, about 30% of the aliphatic protons have been assigned to residue type by two-dimensional spectroscopy. Nuclear Overhauser enhancements establish that high-field methyl groups belonging to a valine residue lie directly over an aromatic ring.     

The primary structure of the DeoR repressor from Escherichia coli K-12.

The nucleotide sequence of the deoR gene of E. coli, which codes for the DeoR repressor, has been determined. This gene codes for a polypeptide that is 252 amino acids residues in length. Computer-assisted analysis of the nucleotide sequence strongly suggests that the DNA binding domain of the DeoR repressor is located in the N-terminal part of the protein. After the coding region there is a dyad symmetry similar to a palindromic unit present outside many structural genes on the E. coli chromosome.     

Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance

The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy. The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical. The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two-state transition. The circular dichroism at 222 nm decreases in two phases as urea is added. Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate. Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content. The second transition is the dissociation and unfolding of the partially unfolded dimer. At high concentrations of protein (500 microM) about 73% of the repressor exists as the intermediate in 4 M urea. The apparent dissociation constant is about 10(-4) M; the subunits are probably strongly stabilised by the subunit interaction. The native repressor is stable up to at least 70 degrees C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature approximately 60 degrees C, delta H approximately 230 kJ/mol).     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of β-galactosidase with symmetric variants of α- and β-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the α-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the α-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for α-centered trp operator variants with exchanges in positions 3, 4 and 5.     

Differential hydration of dA.dT base pairing and dA and dT bulges in deoxyoligonucleotides

The role of water in the formation of stable duplexes of nucleic acids is being studied by determining the concurrent volume change, heats, and counterion uptake that accompany the duplexation process. The variability of the volume contraction that we have observed in the formation of a variety of homoduplexes suggests that sequence and conformation acutely affect the degree of hydration. We have used a combination of densimetric and calorimetric techniques to measure the change in volume and enthalpy resulting from the mixing of two complementary strands to form (a) fully paired duplexes with 10 or 11 base pairs and (b) bulged decameric duplexes with an extra dA or dT unmatched residue. We also monitored absorbance vs temperature profiles as a function of strand and salt concentration for all four duplexes. Relative to the decamer duplex, insertion of an extra dA.dT base pair to form an undecamer duplex results in a favorable enthalpy of -5.6 kcal/mol that is nearly compensated by an unfavorable entropy term of -5.1 kcal/mol. This enthalpy difference correlates with a differential uptake of water molecules, corresponding to an additional hydration of 16 mol of water molecules/mol of base pair. Relative to the fully paired duplexes, both bulged duplexes are 12-16 degrees C less stable and exhibit marginally larger counterion uptake on forming the duplex. The enthalpy change is slightly lower for the T-bulge duplex and less still for the A-bulge duplex. The volume change results indicate that an unmatched residue increases the amount of coulombic and/or structural hydration. The combined results strongly suggest that the destabilizing forces in bulged duplexes are partially compensated by an increase in hydration levels.     

Folding and stability of trp aporepressor from Escherichia coli

Equilibrium and kinetic studies of the urea-induced unfolding of trp aporepressor from Escherichia coli were performed to probe the folding mechanism of this intertwined, dimeric protein. The equilibrium unfolding transitions at pH 7.6 and 25 degrees C monitored by difference absorbance, fluorescence, and circular dichroism spectroscopy are coincident within experimental error. All three transitions are well described by a two-state model involving the native dimer and the unfolded monomer; the free energy of folding in the absence of denaturant and under standard-state conditions is estimated to be 23.3 +/- 0.9 kcal/mol of dimer. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration in the manner expected from the law of mass action for the two-state model. We find no evidence for stable folding intermediates. Kinetic studies reveal that unfolding is governed by a single first-order reaction whose relaxation time decreases exponentially with increasing urea concentration and also decreases with increasing protein concentration in the transition zone. Refolding involves at least three phases that depend on both the protein concentration and the final urea concentration in a complex manner. The relaxation time of the slowest of these refolding phases is identical with that for the single phase in unfolding in the transition zone, consistent with the results expected for a reaction that is kinetically reversible. The two faster refolding phases are presumed to arise from slow isomerization reactions in the unfolded form and reflect parallel folding channels.     

Crystallization of the met repressor from Escherichia coli

The met repressor from Escherichia coli has been crystallized in space group P21, with unit cell dimensions a = 35.6 A, b = 62.6 A, c = 44.5 A, beta = 102.4 degrees and one aporepressor dimer per asymmetric unit. Preliminary X-ray diffraction photographs show measurable intensities to beyond 1.5 A resolution, and the crystal form is ideally suited to high-resolution crystallographic analysis (1 A = 0.1 nm).     

Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes.

DNA binding proteins that induce structural changes in DNA are common in both prokaryotes and eukaryotes. Integration host factor (IHF) is a multi-functional DNA binding and bending protein of Escherichia coli that can mediate protein-protein and protein-DNA interactions by bending DNA. Previously we have shown that the presence of a dA+dT element 5'-proximal to an IHF consensus sequence can affect the binding of IHF to a particular site. In this study the contribution of various sequence elements to the formation of IHF-DNA complexes was examined. We show that IHF bends DNA more when it binds to a site containing a dA+dT element upstream of its core consensus element than to a site lacking a dA+dT element. We demonstrate that IHF can be specifically crosslinked to DNA with binding sites either containing or lacking this dA+dT element. These results indicate the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.     

Sequence-specific NMR assignments of the trp repressor from Escherichia coli using three-dimensional 15N/1H heteronuclear techniques.

Sequence-specific 15N and 1H assignments for the trp holorepressor from Escherichia coli are reported. The trp repressor consists of two identical 107-residue subunits which are highly helical in the crystal state [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L. & Sigler, P. B. (1985) Nature 317, 782-786]. The high helical content and the relatively large size of the protein (Mr = 25,000) make it difficult to assign even the main-chain resonances by conventional homonuclear two-dimensional NMR methods. However, we have now assigned the main-chain resonances of 94% of the residues by using three-dimensional 15N/1H heteronuclear experiments on a sample of protein uniformly labelled with 15N. The additional resolution obtained by spreading out the signals into three dimensions proved indispensable in making these assignments. In particular, we have been able to resolve signals from residues in the N-terminal region of the A helix for the first time in solution. The observed NOE results confirm that the repressor is highly helical in solution, and contains no extended chain conformations.