Lac Repressor DNA-binding Domain Research Articles

Chemical Interactions of Polyethylene Glycols (PEGs) and Glycerol with Protein Functional Groups: Applications to Effects of PEG and Glycerol on Protein Processes.

In this work, we obtain the data needed to predict chemical interactions of polyethylene glycols (PEGs) and glycerol with proteins and related organic compounds and thereby interpret or predict chemical effects of PEGs on protein processes. To accomplish this, we determine interactions of glycerol and tetraEG with >30 model compounds displaying the major C, N, and O functional groups of proteins. Analysis of these data yields coefficients (α values) that quantify interactions of glycerol, tetraEG, and PEG end (-CH2OH) and interior (-CH2OCH2-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with aromatic C, amide N, and cationic N, but unfavorably with amide O, carboxylate O, and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliphatic C are quite favorable, while interactions of glycerol and PEG end groups with aliphatic C are not. Hence, tetraEG and PEG300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD), while glycerol and di- and monoethylene glycol are stabilizers. Favorable interactions with aromatic and aliphatic C explain why PEG400 greatly increases the solubility of aromatic hydrocarbons and steroids. PEG400-steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α values reported here, chemical contributions to PEG m-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA) and separated from excluded volume effects.

Specificity and Affinity of Lac Repressor for the Auxiliary Operators O2 and O3 Are Explained by the Structures of Their Protein–DNA Complexes

The structures of a dimeric mutant of the Lac repressor DNA-binding domain complexed with the auxiliary operators O2 and O3 have been determined using NMR spectroscopy and compared to the structures of the previously determined Lac– O1 and Lac–nonoperator complexes. Structural analysis of the Lac– O1 and Lac– O2 complexes shows highly similar structures with very similar numbers of specific and nonspecific contacts, in agreement with similar affinities for these two operators. The left monomer of the Lac repressor in the Lac– O3 complex retains most of these specific contacts. However, in the right half-site of the O3 operator, there is a significant loss of protein–DNA contacts, explaining the low affinity of the Lac repressor for the O3 operator. The binding mode in the right half-site resembles that of the nonspecific complex. In contrast to the Lac–nonoperator DNA complex where no hinge helices are formed, the stability of the hinge helices in the weak Lac– O3 complex is the same as in the Lac– O1 and Lac– O2 complexes, as judged from the results of hydrogen/deuterium experiments.

Thermal and urea-induced unfolding of the marginally stable lac repressor DNA-binding domain: a model system for analysis of solute effects on protein processes.

Thermodynamic and structural evidence indicates that the DNA binding domains of lac repressor (lacI) exhibit significant conformational adaptability in operator binding, and that the marginally stable helix-turn-helix (HTH) recognition element is greatly stabilized by operator binding. Here we use circular dichroism at 222 nm to quantify the thermodynamics of the urea- and thermally induced unfolding of the marginally stable lacI HTH. Van't Hoff analysis of the two-state unfolding data, highly accurate because of the large transition breadth and experimental access to the temperature of maximum stability (T(S); 6-10 degrees C), yields standard-state thermodynamic functions (deltaG(o)(obs), deltaH(o)(obs), deltaS(o)(obs), deltaC(o)(P,obs)) over the temperature range 4-40 degrees C and urea concentration range 0 </= C(3) </= 6 M. For unfolding the HTH, deltaG(o)(obs) decreases linearly with increasing C(3) at all temperatures examined, which directly confirms the validity of the linear extrapolation method (LEM) to obtain the intrinsic stability of this protein. At 25 degrees C (pH 7.3 and 50 mM K(+)), both linear extrapolation and extrapolation via the local-bulk domain model (LBDM) to C(3) = 0 yield deltaG(o)(obs) = 1.23 +/- 0.05 kcal mol(-)(1), in agreement with direct measurement (1.24 +/- 0.30 kcal mol(-)(1)). Like deltaG(o)(obs), both deltaH(o)(obs) and deltaS(o)(obs) decrease linearly with increasing C(3); the derivatives with respect to C(3) of deltaG(o)(obs), deltaH(o)(obs) and TdeltaS(o)(obs) (in cal mol(-)(1) M(-)(1)) are -449 +/- 11, -661 +/- 90, and -203 +/- 91 at 25 degrees C, indicating that the effect of urea on deltaG(o)(obs) is primarily enthalpic. The deltaC(o)(P,obs) of unfolding (0.63 +/- 0.05 kcal mol(-)(1) K(-)(1)) is not detectibly dependent on C(3) or temperature. The urea m-value of the lacI HTH (-d deltaG(o)(obs),/dC(3) = 449 +/- 11 cal mol(-)(1) M(-)(1) at 25 degrees C) is independent of C(3) up to at least 6 M. Use of the LBDM to fit the C(3)-dependence of deltaG(o)(obs) yields the local-bulk partition coefficient for accumulation of urea at the protein surface exposed upon denaturation: K(P) = 1.103 +/- 0.002 at 25 degrees C. This partition coefficient is the same within uncertainty as those previously determined by LBDM analysis of osmometric data for solutions of urea and native (folded) bovine serum albumin, as well as LBDM analysis of the proportionality of m-values to changes in water accessible surface area upon protein unfolding. From the correspondence between values of K(P), we conclude that the average local urea concentration at both folded and unfolded protein surface exceeds the bulk by approximately 10% at 25 degrees C. The observed decrease in m-value for the lacI HTH with increasing temperature, together with the observed reductions in both deltaH(o)(obs) and deltaS(o)(obs) of unfolding with increasing urea concentration, demonstrate that K(P) for urea decreases with increasing temperature and that transfer of urea from the bulk solution to the local domain at the protein surface exposed on denaturation is enthalpically driven and entropically unfavorable.

Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.

The lac repressor-operator system is a model system for understanding protein-DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the specific recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the first high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator 01. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48 degrees of the right lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor-operator system is unique among other protein-DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.

Wrapping of flanking non-operator DNA in lac repressor-operator complexes: implications for DNA looping

In our studies of lac repressor tetramer (T)-lac operator (O) interactions, we observed that the presence of extended regions of non-operator DNA flanking a single lac operator sequence embedded in plasmid DNA produced large and unusual cooperative and anticooperative effects on binding constants (Kobs) and their salt concentration dependences for the formation of 1:1 (TO) and especially 1:2 (TO2) complexes. To explore the origin of this striking behavior we report and analyze binding data on 1:1 (TO) and 1:2 (TO2) complexes between repressor and a single Osym operator embedded in 40 bp, 101 bp, and 2514 bp DNA, over very wide ranges of [salt]. We find large interrelated effects of flanking DNA length and [salt] on binding constants (KobsTO, KobsTO2) and on their [salt]-derivatives, and quantify these effects in terms of the free energy contributions of two wrapping modes, designated local and global. Both local and global wrapping of flanking DNA occur to an increasing extent as [salt] decreases. Global wrapping of plasmid-length DNA is extraordinarily dependent on [salt]. We propose that global wrapping is driven at low salt concentration by the polyelectrolyte effect, and involves a very large number (≳20) of coulombic interactions between DNA phosphates and positively charged groups on lac repressor. Coulombic interactions in the global wrap must involve both the core and the second DNA-binding domain of lac repressor, and result in a complex which is looped by DNA wrapping. The non-coulombic contribution to the free energy of global wrapping is highly unfavorable (∼+30-50 kcal mol−1), which presumably results from a significant extent of DNA distortion and/or entropic constraints. We propose a structural model for global wrapping, and consider its implications for looping of intervening non-operator DNA in forming a complex between a tetrameric repressor (LacI) and one multi-operator DNA molecule in vivo and in vitro. The existence of DNA wrapping in LacI-DNA interactions motivates the proposal that most if not all DNA binding proteins may have evolved the capability to wrap and thereby organize flanking regions of DNA.

The solution structure of Lac repressor headpiece 62 complexed to a symmetrical lac operator

Lactose repressor protein (Lac) controls the expression of the lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the lac operon. Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator. Protein-protein and protein-DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail. We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized lac operator. Analysis of the structures reveals specific interactions between Lac repressor and DNA that were not found in previously investigated Lac repressor-DNA complexes. Important differences with the previously reported structures of the HP56-DNA complex were found in the loop following the helix-turn-helix (HTH) motif. The protein-protein and protein-DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail. The structures were also used for comparison with the available crystallographic data on the Lac and Pur repressor-DNA complexes. The structures of the HP62-DNA complex provide the basis for a better understanding of the specific recognition in the Lac repressor-operator complex. In addition, the structural features of the hinge region provide detailed insight into the protein-protein and protein-DNA interactions responsible for the high affinity of the repressor for operator DNA.

Refined Structure of lacRepressor Headpiece (1-56) Determined by Relaxation Matrix Calculations from 2D and 3D NOE Data: Change of Tertiary Structure upon Binding to the lacOperator

The solution structure of the DNA binding domain of lacrepressor (headpiece 1-56; HP56) has been refined using data from 2D and 3D NMR spectroscopy. The structure was derived from 1546 restraints (giving an average of 27.6 per residue), comprising 389 intraresidual, 402 sequential, 385 medium range and 325 long range distance restraints and also 30 φ and 15 χ 1dihedral angle restraints. The structures were determined by the method of direct refinement against nuclear Overhauser enhancement peak volumes with the program DINOSAUR. The final set of 32 selected structures displayed an r.m.s. deviation from the average of 0.43(±0.08) Å (backbone) and 0.95(±0.08) Å (all heavy atoms) for the best defined region of the protein (residues 3 to 49). The ensemble R-factor was 0.35, which indicates close correspondence with the experimental data. The structures revealed good stereochemical qualities. The conformations of the NMR structures of free and DNA complexed lacrepressor headpiece were compared. The regions comprising the secondary structure elements show close correspondence for both conformations. However, the conformation of the loop between helix II and III changes considerably upon complexation of the headpiece. This change in the conformation of the loop in lacHP56 is essential for binding of the side-chains of residues Asn25 and His29 to the lacoperator DNA. Finally, the lacheadpiece residues that are intolerant to mutations were analysed. Most of these mutation-sensitive residues are important for a correct folding of the headpiece region, and a number of these residues are also involved in contacting the operator DNA.

Mechanism of Lac repressor switch-off: orientation of the Lac repressor DNA-binding domain is reversed upon inducer binding

Lac repressor's DNA-binding domains contain helix-turn-helix motif which, though similar to those of phage λ Cro protein, are oriented differently with respect to DNA: in the specific complexes with Lac operator, N termini of the repressor's subunits are facing inwards. We demonstrate that, in the presence of an inducer, the repressor's N termini cross-link to the operator's outermost nucleotides. We suggest that the inducer fixes the repressor's DNA-binding domains in the Cro-type configuration and thus garbles its recognition surface. Since the Cro-type configuration is perfectly suitable for binding the DNA, this also explains how the switched-off repressor retains its non-specific DNA-binding.

Orientation of the Lac repressor DNA binding domain in complex with the leftlacoperator half site characterized by affinity cleaving

Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein. Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP [Boelens, R., Scheek, R., van Boom, J. and Kaptein, R., J. Mol. Biol. 193, 1987, 213-216]. In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method. The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR. We have formed the complex between the modified protein and the left DNA half site for LacR. The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.

Combined procedure of distance geometry and restrained molecular dynamics techniques for protein structure determination from nuclear magnetic resonance data: application to the DNA binding domain of lac repressor from Escherichia coli.

The technique of two-dimensional nuclear magnetic resonance (2D-NMR) has recently assumed an active role in obtaining information on structures of polypeptides, small proteins, sugars, and DNA fragments in solution. In order to generate spatial structures from the atom-atom distance information obtained by the NMR method, different procedures have been developed. Here we introduce a combined procedure of distance geometry (DG) and molecular dynamics (MD) calculations for generating 3D structures that are consistent with the NMR data set and have reasonable internal energies. We report the application of the combined procedure on the lac repressor DNA binding domain (headpiece) using a set of 169 NOE and 17 "hydrogen bond" distance constraints. Eight of ten structures generated by the distance geometry algorithm were refined within 10 ps MD simulation time to structures with low internal energies that satisfied the distance constraints. Although the combination of DG and MD was designed to combine the good sampling properties of the DG algorithm with an efficient method of lowering the internal energy of the molecule, we found that the MD algorithm contributes significantly to the sampling as well.

Interactive program for investigation of protein structures based on 1H NMR experiments

An interactive molecular graphics program running on an Evans & Sutherland picture system is presented. It allows work on the conformations of a maximum of five molecules with a combined total of up to 1 000 atoms. Conformation changes are executed by real-time variations of dihedral angles about single bonds. Up to eight torsion angles can be selected anywhere in the molecule and changed simultaneously by turning dials. The program aims to establish geometric structures of molecules based on conformation-dependent 1H NMR data; primarily upper limits on distances between protons. These may be located anywhere in the sequence of amino acids, which requires that the user is given access to the whole structure, i.e. all atoms and all dihedral angles, at any time. Violations of the upper limits in the actual conformations are displayed graphically to guide the user in search of conformations consistent with the 1H NMR data. Applications discussed include various aspects of the previously published 1 determination of an approximate, topologically correct structure of the lac repressor DNA-binding domain from E. coli, and interactive steps in the energy refinement of the solution conformation of a protease inhibitor from bull seminal plasma, which was obtained from distance geometry calculations with NMR data. Furthermore, the present graphics program proved to be a useful tool for detailed examinations and comparisons of protein structures obtained either with crystallographic methods or with the combined use of NMR and distance geometry.

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.

Sequence-specific resonance assignments in the 1H nuclear-magnetic-resonance spectrum of the lac repressor DNA-binding domain 1-51 from Escherichia coli by two-dimensional spectroscopy.

The assignment of the 1H nuclear magnetic resonance (NMR) spectrum of the DNA-binding domain 1-51 of lac repressor from Escherichia coli is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional NMR experiments at 360 MHz and 500 MHz. Individual assignments were obtained at 18 degrees C for the backbone protons of 44 out of the total of 51 amino acids residues, the exceptions being Met-1, Lys-2, Tyr-7, Arg-35, Glu-36, Lys-37 and Ile-48. Complete assignments of the non-labile hydrogen atoms of the side chain were obtained for 33 residues, and for Asn-46 and Asn-50 the delta amide protons were also identified. The chemical shifts for the assigned resonances at 18 degrees C are listed for an aqueous solution at pH 4.9 and at pH 6.8.

Secondary structure of the lac repressor DNA-binding domain by two-dimensional 1H nuclear magnetic resonance in solution.

A recently proposed approach for spatial structure determination in noncrystalline proteins by nuclear magnetic resonance was applied to the lac repressor DNA-binding domain. On the basis of sequence-specific 1H NMR assignments, the location of alpha-helices in the amino acid sequence was determined from nuclear Overhauser enhancement data and from amide proton exchange studies. These investigations provide detailed experimental data on the structure of a noncrystalline DNA-binding protein. The results support the hypothesis advanced by others that sequence-specific interactions between lac repressor and DNA are mediated by a particular spatial arrangement of two alpha-helices common to various different DNA-binding proteins.

Photochemically induced dynamic nuclear polarization investigation of complex formation of the NH2-terminal DNA-binding domain of lac repressor with poly[d(AT)

The interaction of the NH2-terminal DNA-binding domain of lac repressor with synthetic oligo[d(AT)] was studied by a photo-CIDNP technique (CIDNP is chemically induced dynamic nuclear polarization). Three of the four tyrosines of the NH2-terminal region were found to be accessible to the photosensitive dye. The corresponding ring proton resonances were enhanced in the photo-CIDNP 1H NMR spectrum, and the only histidine (histidine 29) was located at the surface of the domain, which is supposed to be linked to the core protein of lac repressor by a flexible hinge region. After complex formation of the NH2-terminal region with oligo[d(AT)], two of the three tyrosine residues were no longer accessible to solvent or to photosensitive dye, which is strong evidence that the two tyrosines are part of the contact region.