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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.     

Structure of a variant of lac repressor with increased thermostability and decreased affinity for operator

A single amino acid substitution, K84L, in the Escherichia coli lac repressor produces a protein that has substantially increased stability compared to wild-type. However, despite the increased stability, this altered tetrameric repressor has a tenfold reduced affinity for operator and greatly decreased rate-constants of inducer binding as well as a reduced phenotypic response to inducer in vivo. To understand the dramatic increase in stability and altered functional properties, we have determined the X-ray crystal structures of a dimeric repressor with and without the K84L substitution at resolutions of 1.7 and 3.0 A, respectively. In the wild-type dimer, K84-11, Lys84 forms electrostatic interactions at the monomer-monomer interface and is partially exposed to solvent. In the K84L-11 substituted protein there is reorientation of the N-subdomains, which allows the leucine to become deeply buried at the monomer-monomer interface. This reorientation of the N-subdomains, in turn, results in an alteration of hydrogen bonding, ion pairing, and van der Waals interactions at the monomer-monomer interface. The lysine residue at position 84 appears to exert its key effects by destabilizing the "optimal" conformation of the repressor, effectively loosening the dimer interface and allowing the repressor to adopt the conformations necessary to function as a molecular switch.     

Effects of dominant-negative lac repressor mutations on operator specificity and protein stability

The specific binding of dominant-negative (I-d) lactose (lac) repressors to wild-type (wt) as well as mutant (Oc) lac operators has been examined to explore the sequence-specific interaction of the lac repressor with its target. Mutant lacI genes encoding substitutions in the N-terminal 60 amino acids (aa) were cloned in a derivative of plasmid pBR322. Twelve of these lacI-d missense mutations were transferred from F'lac episomes using general genetic recombination and molecular cloning, and nine lacI missense mutations were recloned from M13-lacI phages [Mott et al., Nucl. Acids Res. 12 (1984) 4139-4152]. The mutant repressors were examined for polypeptide size and stability, for binding the inducer isopropyl-beta-D-thiogalactoside (IPTG), as well as binding to wt operator. The mutant repressors' affinities for wt operator ranged from undetectable to about 1% that of wt repressor, and the mutant repressors varied in transdominance against repressor expressed from a chromosomal lacIq gene. Six of the I-d repressors were partially degraded in vivo. All repressors bound IPTG with approximately the affinity of wt repressor. Repressors having significant affinity for wt operator or with substitutions in the presumed operator recognition helix (aa 17-25) were examined in vivo for their affinities for a series of single site Oc operators. Whereas the Gly-18-, Ser-18- and Leu-18-substituted repressors showed altered specificity for position 7 of the operator [Ebright, Proc. Natl. Acad. Sci. USA 83 (1986) 303-307], the His-18 repressor did not affect specificity. This result may be related to the greater side-chain length of histidine compared to the other amino acid substitutions.     

Tight-binding repressors of the lac operon: selection system and in vitro analysis.

The isolation and characterization of altered repressors of the lac operon which have an increased affinity for an operator should give useful clues about the molecular basis for the very tight and specific interaction between repressor and operator. A selection system has been devised which allows the isolation of such repressor mutants. This system selects for mutant repressors which can overcome lac operator-constitutive (Oc) mutations. By using in vivo assays, 24 candidates were obtained which, compared with wild type, have an increased trans effect of their repressor on one or several Oc operators. Three of these candidates have been investigated in vitro; the affinity of their repressor for inducer was unchanged, whereas the affinity for wild-type operator was increased 15-, 86-, and 262-fold, respectively.     

Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.

We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor.     

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids.

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

Evidence for leucine zipper motif in lactose repressor protein

Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.     

Ultraviolet radiation-induced derepression of the lactose operon of E. coli

The effect of ultraviolet irradiation of a regulatory protein, the lac repressor, on its interactions with operator DNA is investigated by spectroscopic and electrophoresis methods. A second set of experiments is performed to assay the capacity of the system containing the irradiated repressor to be induced by IPTG. The protein-nucleic acid interactions are modified upon ultraviolet irradiation of the repressor. The inducer becomes ineffective and repressor stays "locked" to DNA in conditions in which the native repressor is released from the system. These facts are discussed in terms of genes repression and of promotion step in ultraviolet induced carcinogenesis.     

Specific binding of lac repressor to linear versus circular polyoperator molecules

Gel shift assays were used to examine the binding of the lactose (lac) repressor to polyoperator DNA molecules. Specific binding was differentiated from nonspecific DNA association by (i) equilibrating repressor-operator complexes below the nonspecific association constant and (ii) demonstrating the effects of the inducer isopropyl beta-D-thiogalactoside (IPTG) on the formation of repressor-operator complexes. With the linear polyoperator molecules, all eight operator sites could be simultaneously bound by distinct repressors. However, with circular molecules, the eight operator sites were saturable by repressor only in the nicked circular state and not in the covalently closed circular form. Under the experimental conditions used, there was no evidence of bifunctional repressor binding or loop formation. The results suggest that the conformational perturbation of DNA that occurs upon specific repressor binding was retained in topologically closed molecules and could modify other operator sites so as to make them unavailable for specific binding.     

Detection of protein-DNA interaction and regulation using gold nanoparticles

The interaction between protein and DNA is usually regulated by a third species, an effector, which can be either a protein or a small molecule. Convenient methods capable of detecting protein-DNA interaction and its regulation are highly desirable research tools. In the current study, we developed a method to directly “visualize” the interaction between a protein-DNA pair and its effector through the coupling with gold nanoparticles (AuNPs). As a proof-of-concept experiment, we constructed a model system based on the interaction between the lac repressor (protein) and operator (DNA) and its interplay with the lac operon inducer isopropyl β-d-1-thiogalactopyranoside (IPTG, which inhibits the interaction between the lac repressor and operator). We coated AuNPs with the lac operator sequences and mixed them with the lac repressor. Because the lac repressor homotetramer contains two DNA binding modules, it bridged the particles and caused them to aggregate. We demonstrated that the assembly of DNA-modified AuNPs correlated with the presence of the corresponding protein and effector in a concentration-dependent manner. This AuNP-based platform has the potential to be generalized in the creation of reporter and detection systems for other interacting protein-DNA pairs and their effectors.     

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.     

Tissue-Specific Distribution of DNA-Binding Nuclear Proteins

Abstract. The DNA-binding capacity of nuclear proteins of mouse cells was examined by the protein-blotting method. Under conditions in which the lac repressor specifically binds to the lac operator, the DNA-binding nuclear proteins from different tissues showed a tissue-specific distribution, suggesting that the species and amounts of nuclear proteins with DNA binding activity differ in different tissues.
When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor.     

Tissue-specific distribution of DNA-binding nuclear proteins

The DNA-binding capacity of nuclear proteins of mouse cells was examined by the protein-blotting method. Under conditions in which the lac repressor specifically binds to the lac operator, the DNA-binding nuclear proteins from different tissues showed a tissue-specific distribution, suggesting that the species and amounts of nuclear proteins with DNA binding activity differ in different tissues. When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor.