lac Repressor headpiece binds specifically to half of the lac operator: a proton nuclear magnetic resonance study

The complex formation of the N-terminal domain (headpiece) of the Escherichia coli lac repressor and a synthetic 14-base-pair lac operator fragment has been investigated by 1H NMR. Titration shifts in the imino-proton region of the DNA spectrum and in the aromatic region of the headpiece spectrum are examined in detail and interpreted where possible. The assignment of the resonances in the complex follows in part from the titration data and is completed by nuclear Overhauser measurements. The shift of the His-29 C-2 resonance has been used to assess the binding strength of the complex. Evidence is presented for the presence of a high-affinity site on the lac operator fragment (KD less than or equal to 2 X 10(-5) M), which shows features in common with one of the specific binding sites on the complete lac operator, and for the presence of a second, nonspecific binding site with lower affinity. The influence of this second site on the interpretation of the binding data is discussed.     

A protein structure from nuclear magnetic resonance data. lac repressor headpiece

A procedure is described to determine the three-dimensional structure of biomolecules from nuclear magnetic resonance data. This procedure combines model building with a restrained molecular dynamics algorithm, in which distance information from nuclear Overhauser effects is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or headpiece (amino acid residues 1 to 51) of the lac repressor from Escherichia coli, for which no crystal structure is available. The relative orientation of the three helices of the headpiece is similar to that of the three homologous helices found in the cI repressor of bacteriophage lambda.     

H NMR study of a complex between the lac repressor headpiece and a 22 base pair symmetric lac operator

A complex between the lac repressor headpiece and a fully symmetric tight-binding 22 bp lac operator was studied by 2D NMR. Several 2D NOE spectra were recorded for the complex in both H2O and 2H2O. Many NOE cross-peaks between the headpiece and DNA could be identified, and changes in the chemical shift of the DNA protons upon complex formation were analyzed. Comparison of these data with those obtained for a complex between the headpiece and a 14 bp half-operator, studied previously [Boelens, R., Scheek, R. M., Lamerichs, R. M. J. N., de Vlieg, J., van Boom, J. H., & Kaptein, R. (1987) in DNA-ligand interactions (Guschlbauer, W., & Saenger, W., Eds.) pp 191-215, Plenum, New York], shows that two headpieces form a specific complex with the 22 bp lac operator in which each headpiece binds in the same way as found for the 14 bp complex. The orientation of the recognition helix in the major groove of DNA in these complexes is opposite with respect to the dyad axis to that found for other repressors.     

NMR study of the interaction between the lac repressor and the lac operator

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few imino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form.     

Two-dimensional 1H, 15N NMR investigation of uniformly 15N-labeled lac repressor headpiece.

15N uniformly labeled lac repressor and lac repressor headpiece were prepared. 15N NMR spectra of lac repressor were shown resolution inadequate for detailed study while the data showed that the 15N labeled N-terminal part of the protein is quite suitable for this type of study allowing future investigation of the specific interaction of the lac repressor headpiece with the lac operator. We report here the total assignment of proton 1H and nitrogen 15NH backbone resonances of this headpiece in the free state. Assignments of the 15N resonances of the protein were obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC nuclear Overhauser and relayed HMQC-HOHAHA spectroscopy. More than 80 per cent of residues were assigned by their 15NH(i)-N1H(i + 1) and 15NH(i)-N1H(i - 1) connectivities. Values of the 3JNH alpha splitting for 39 of the 51 residues of the headpiece were extracted from HMQC and HMQC-J. The observed 15NH(i)-C beta H cross peaks and the 3JNH alpha coupling constants values are in agreement with the three alpha-helices previously described [Zuiderweg, E.R.P., Scheek, R.M., Boelens, R., van Gunsteren, W.F. and Kaptein, R., Biochimie 67, 707 (1985)]. The 3JNH alpha coupling constants can be now used for a more confident determination of the lac repressor headpiece. From these values it is shown that the geometry of the ends of the second and third alpha-helices exhibit deviation from the canonical alpha-helix structure. On the basis of NOEs and 3JNH alpha values, the geometry of the turn of the helix-turn-helix motif is discussed.     

Interaction between the lac operator and the lac repressor headpiece: fluorescence and circular dichroism studies

The interaction between the lac repressor headpiece and a small operator DNA fragment has been examined by fluorescence and circular dichroism (c.d.) measurements. Binding of the headpiece to the DNA fragment induces a strong quenching of the fluorescence of its tyrosine residues. Quantitative analysis of the fluorescence data demonstrates that, in a first step, two headpieces bind very strongly to the DNA fragment then weaker binding occurs. C.d. demonstrates that the binding induces conformational changes of the DNA. The c.d. change produced upon binding of the first two headpieces differs from that induced upon binding of two further headpieces . Binding of the second pair of headpieces is similar to non-specific binding to non-operator DNA. The conformation of the operator DNA in the presence of two headpieces differs drastically from that in presence of lac repressor. Addition of the core to the lac operator does not induce any conformational change of the nucleic acids. These results are discussed with respect to the relative roles of core and headpieces in the lac repressor-lac operator interaction.     

An NMR-based molecular dynamics simulation of the interaction of the lac repressor headpiece and its operator in aqueous solution

The results of a 125 psec molecular dynamics simulation of a lac headpiece-operator complex in aqueous solution are reported. The complex satisfies essentially all experimental distance information derived from two-dimensional nuclear magnetic resonance (2-D-NMR) studies. The interaction between lac repressor headpiece and its operator is based on many direct- and water-mediated hydrogen bonds and nonpolar contacts which allow the formation of a tight complex. No stable hydrogen bonds between side chains and bases are found, while specific contacts occur between both nonpolar groups and, to a lesser extent, through water-mediated hydrogen bonds. The simulated complex structure in water is intrinsically stable without application of nuclear Overhauser effect (NOE) distance restraints, while being compatible with most of the available biochemical, genetic, and chemically induced dynamic nuclear polarization (CIDNP) data.     

Photo-CIDNP study of the interaction between lac repressor headpiece and lac operator DNA

Lac repressor headpiece (HP) and intact lac repressor have been studied using the photo-CIDNP method. At neutral pH histidine 29, tyrosines 7, 12 and 17 and methionine 1 are polarised. His-29 polarizations are weaker and broader in HP59 than in HP51 indicating that the C-terminal octapeptide in HP59 adopts a conformation that allows an interaction with His-29. The photo-CIDNP spectra of intact lac repressor and HP51 are very similar, showing that the same residues are accessible to the photo-excited flavin. An equimolar mixture of HP51 and a 14 base pair lac operator fragment strongly suppresses the photo-CIDNP effect of tyrosines 7 and 17 and abolishes the His-29 polarizations. The results are compared with earlier photo-CIDNP measurements on a complex of headpiece with poly[d(AT)] and with a model derived from a 2D NMR study on a lac headpiece-operator complex.     

Assignment of the 1H-NMR spectrum of a lac repressor headpiece-operator complex in H2O and identification of NOEs. Consequences for protein-DNA interaction

A complex between the headpiece amino-terminal residues 1-56 of lac repressor (HP56) and an 11-bp lac operator fragment was studied by 1H NMR. The sequence specific assignment of the exchangeable and non-exchangeable protons has been accomplished. Several protons have favourable chemical shifts in the complex, therefore new intraprotein NOEs could be found that had not been unambigously identified in the free protein. By comparison, most of these intraprotein NOEs are also present in the spectra of the free headpiece but some are different. Furthermore, several new proteins DNA NOEs could be identified. The NOE between the side-chain amide protons of Gln18 and C5H of C7 confirms the specific contact between these residues which was proposed from genetic experiments [Ebright, R. M. (1985) J. Biomol. Struct. & Dyn. 3, 281-297]. The implications of the new data for the interaction between the lac repressor headpiece and its operator are discussed.     

Structure of the complex between lac repressor headpiece and operator DNA from measurements of the orientation relaxation and the electric dichroism.

The complex between lac repressor headpiece and short rodlike DNA fragments containing the lac operator sequence is characterised by measurements of the rotation diffusion. Using the method of electric dichroism we measure the rotation relaxation and determine changes in the length of the DNA upon ligand binding with high accuracy. According to these measurements any change in the length of the operator DNA upon binding of the first two headpiece molecules remains below 1A; the electric dichroism also remains virtually unchanged. At high degrees of (unspecific) binding we observe an increase in the rotation relaxation time, which is attributed to an increase of the apparent mean radius of the complex. As a control of our procedure for the determination of length changes we use the intercalation of ethidium bromide and arrive at an increase of the DNA length per bound ethidium of 3.2A (at 3.4A rise per base pair). The results obtained for the headpiece operator complex are not consistent with models assuming large changes of the DNA structure or intercalation of tyrosine residues.     

Energetics of interaction of oligopeptide (lac 53-57) with DNA base sequences and origin of sequence-specific recognition

Computer model building with a dynamic energy minimization procedure is used here to study the interaction of a pentapeptide sequence from the lac repressor headpiece (lac 53-57) with different base sequences of DNA. The peptide fragment for this purpose was considered in the classical beta-antiparallel as well as the beta-associated conformation. The model of its interaction with DNA was optimised for various binding positions and base sequences. Partitioning of energy is analysed for different dielectric constant values and the main contributing factors to sequence-specific binding are discussed.     

31P NMR spectra of an oligodeoxyribonucleotide duplex lac operator-repressor headpiece complex

The interaction of a symmetric lac operator duplex, d(TGTGAGCGCTCACA)2, with the N-terminal 56-residue headpiece fragment of the lac repressor protein was monitored by 31P NMR spectroscopy. The changes in the 31P chemical shifts upon addition of the headpiece demonstrated an end point of two headpiece fragments per symmetric 14-mer duplex with each headpiece binding to the T1pG2pT3pG4pA5 ends of the duplex. The specific phosphate 31P perturbations observed are consistent with those residues implicated in protein binding by previous NMR, molecular biological, and biochemical techniques. Upon complexation, the 31P signals of phosphates G2-A5 showed upfield or downfield shifts (less than 0.2 ppm) while most other residues were unperturbed. The interactions were dependent on ionic strength. The 31P NMR data provide direct evidence for predominant recognition of the 5' strand of the 5'-TGTGA/3'-ACACT binding site.     

DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor.

We have used a gel retardation assay to investigate the influence of DNA supercoiling on loop formation between lac repressor and two lac operators. A series of 15 DNA minicircles of identical size (452 bp) was constructed carrying two lac operators at distances ranging from 153 to 168 bp. Low positive or negative supercoiling (sigma = +/- 0.023) changed the spacing between the two lac operators required for the formation of the most stable loops. This reveals the presence of altered double helical repeats (ranging from 10.3 to 10.7 bp) in supercoiled DNA minicircles. At elevated negative supercoiling (sigma = -0.046) extremely stable loops were formed at all operator distances tested, with a slight spacing periodicity remaining. After relaxation of minicircle-repressor complexes with topoisomerase I one superhelical turn was found to be constrained in those minicircles which carry operators at distances corresponding to a non-integral number of helical turns. This indicates that DNA loop formation can define local DNA domains with altered topological properties of the DNA helix.     

Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: the lac repressor DNA binding domain

A procedure is described to determine from NMR data the three-dimensional structure of biomolecules. This procedure combines model building with a restrained Molecular Dynamics algorithm, in which distance information from NOEs is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or "headpiece" (amino acids 1-51) of the lac repressor from E. coli, for which no crystal structure is available. The spatial structure of the headpiece is discussed in terms of known physical and biochemical data and of its DNA binding properties.     

Cooperative alpha-helix unfolding in a protein-DNA complex from hydrogen-deuterium exchange

We present experimental evidence for a cooperative unfolding transition of an alpha-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations.     

The interaction of the recognition helix of lac repressor with lac operator.

We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.     

Spatial arrangement of the three alpha helices in the solution conformation of E. coli lac repressor DNA-binding domain

The relative orientations of the 3 helices in the DNA-binding domain ('headpiece') of lac repressor have been determined using distance constraints obtained from 2-dimensional 1H nuclear Overhauser enhancement spectra. The relative orientations of its helices is similar to that of the central 3 helices in the DNA-binding domain of the lambda repressor of the bacteriophage lambda.     

Mutant lac repressors with new specificities hint at rules for protein--DNA recognition.

Proteins which recognize specific sequences of DNA play a fundamental role in the regulation of protein synthesis in all organisms. A particular helix of the bacterial protein lac repressor recognizes the bases in the major groove of the lac operator. We show that the first two residues of this recognition helix interact independently with two base pairs. This allows us in many cases to predict repression as an indicator of strength of the repressor-operator complex. Rules of recognition can be derived for 16 symmetric operators. They also apply to the gal repressor and possibly to other bacterial repressors.     

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.     

The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding

We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix. We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator. We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix. Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator. They further suggest that residue 9 of the recognition helix (asparagine 25) interacts non-specifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3.