Mutation in hinge region of lactose repressor protein alters physical and functional properties

A mutant of the Escherichia coli lactose repressor (BG124) in which serine at position 77 is replaced by leucine has been examined by physical methods. Consistent with the phenotypic character of this i-d mutant, BG124 protein did not bind lactose operator specifically, but did bind to DNA nonspecifically. Titration with inducer monitoring tryptophan fluorescence changes yielded a biphasic saturation curve, and Scatchard and Hill plots of the fluorescence and equilibrium dialysis data demonstrated heterogeneity of inducer binding sites. Although ultraviolet difference spectra and potassium iodide quenching of fluorescence indicated that BG124 repressor has structural distinctions from wild-type protein, circular dichroism spectra and acrylamide quenching of fluorescence for the two proteins were quite similar. A significantly greater increase of 1-anilino-8-naphthalenesulfonate fluorescence was observed in the presence of mutant versus wild-type repressor. Unlike wild-type behavior, changes in both 1-anilino-8-naphthalenesulfonate fluorescence intensity and maximum emission wavelength in response to inducer were found for the BG124 protein. These results are consistent with conformational alterations in the interface between NH2-terminal and core domains of this mutant repressor. The single amino acid alteration in the hinge between the core and NH2 terminus yields conformational effects which influence physical and functional properties associated with both domains.     

Operator DNA sequence variation enhances high affinity binding by hinge helix mutants of lactose repressor protein

The mechanism by which genetic regulatory proteins discern specific target DNA sequences remains a major area of inquiry. To explore in more detail the interplay between DNA and protein sequence, we have examined binding of variant lac operator DNA sequences to a series of mutant lactose repressor proteins (LacI). These proteins were altered in the C-terminus of the hinge region that links the N-terminal DNA binding and core sugar binding domains. Variant operators differed from the wild-type operator, O(1), in spacing and/or symmetry of the half-sites that contact the LacI N-terminal DNA binding domain. Binding of wild-type and mutant proteins was affected differentially by variations in operator sequence and symmetry. While the mutant series exhibits a 10(4)-fold range in binding affinity for O(1) operator, only a approximately 20-fold difference in affinity is observed for a completely symmetric operator, O(sym), used widely in studies of the LacI protein. Further, DNA sequence influenced allosteric response for these proteins. Binding of this LacI mutant series to other variant operator DNA sequences indicated the importance of symmetry-related bases, spacing, and the central base pair sequence in high affinity complex formation. Conformational flexibility in the DNA and other aspects of the structure influenced by the sequence may establish the binding environment for protein and determine both affinity and potential for allostery.     

Extrinsic interactions dominate helical propensity in coupled binding and folding of the lactose repressor protein hinge helix

A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short alpha-helices when bound to DNA but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge-helix folding and/or hinge-hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality.     

a1 protein alters the DNA binding specificity of alpha 2 repressor

The alpha 2 protein of S. cerevisiae, the product of the MAT alpha 2 gene, represses a set of cell-type-specific genes (the a-specific genes) by binding to an operator sequence upstream of each gene. We demonstrate that a second yeast regulatory protein, a1, the product of the MATa1 gene, can alter the binding specificity of alpha 2 so that it no longer recognizes the a-specific gene operator, but instead acquires the ability to recognize a different operator sequence found upstream of haploid-specific genes. Thus, under the influence of a1, alpha 2 can repress haploid-specific genes. An alpha cell expresses alpha 2 but not a1, so that alpha 2 turns off only the a-specific genes. An a/alpha cell makes both a1 and alpha 2, in a ratio that ensures that alpha 2 is distributed between two distinct binding modes: the alpha 2 binding mode and the a1-alpha 2 binding mode. Thus in an a/alpha cell, alpha 2 represses two distinct classes of genes.     

Tryptic core protein of lactose repressor binds operator DNA.

The core protein produced by mild proteolytic digestion of lactose repressor protein has been purified from native repressor by chromatography on phosphocellulose. The core protein isolated in this manner binds to operator DNA with an apparent dissociation constant of 10(-7) M, and the observed binding is decreased by the presence of inducer. Competition studies with nonspecific DNA indicate that the binding species in the core protein preparations is neither intact lactose repressor nor mixed tetramers containing varying numbers of intact NH2-terminal regions. This conclusion is supported by experiments designed to measure the rate of dissociation of the core protein from the operator DNA. Calculations based on the assumption that the isolated core protein binds similarly to the corresponding region in intact repressor protein indicate that the core region contributes approximately 40 to 50% of the energy of binding to operator DNA. Furthermore, the change in operator affinity upon inducer binding to core accounts for a minimum of 60% of the free energy change in binding to operator observed for the native protein. The demonstration that core protein binds to operator DNA requires a re-evaluation of the various models for repressor binding to DNA. A possible model based on the available information is presented.     

Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding

Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.     

Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding.

lac repressor can be dissected by trypsin into a homogenous tetrameric core (accounting for residues 60 to 347), carrying inducer binding activity, and the monomeric amino-terminal peptides ("headpieces") accounting for residues 1 to 59 and 1 to 51, respectively. This restriction of the action of trypsin on lac repressor is obtained in 1 M Tris-HCl (pH 7.5)-30% in glycerol at 25 degrees C since only the peptide bonds at lysine-59 and to a lesser extent after at arginine-51 are cleaved under these conditions. The headpieces can be purified by gel filtration. They have ordered secondary structure as revealed by circular dichroism studies. The monomeric headpieces show the relatively weak binding to nonoperator DNA but not the highly specific and strong binding to operator DNA typical for tetrameric lac repressor.     

Engineered disulfide linking the hinge regions within lactose repressor dimer increases operator affinity, decreases sequence selectivity, and alters allostery.

The hinge domain encompasses amino acids 51-60 of lactose repressor (LacI) and plays an important role in its regulatory interaction with operator DNA. This segment makes both hinge-DNA and hinge-hinge' contacts that are critical to DNA binding. Furthermore, this small region serves as a central element in communicating the allosteric response to inducer. Introducing a disulfide bond between partner hinges within a dimer via the mutation V52C results in a protein that has increased affinity for O(1) operator DNA compared to wild-type LacI and abolishes allosteric response to inducer [Falcon, C. M., Swint-Kruse, L., and Matthews, K. S. (1997) J. Biol. Chem. 272, 26818]. We have established that this high affinity is maintained for the disulfide-linked protein even when symmetry and half-site spacing within the operator region are altered, whereas binding by the reduced protein, as for wild-type LacI, is severely diminished by these alterations. Interestingly, the allosteric response to inducer for V52C-oxidized remains intact for a small group of operator variants. Temperature studies demonstrate that the presence of the disulfide alters the thermodynamics of the protein-DNA interaction, with a DeltaC(p) of significantly smaller magnitude compared to wild-type LacI. The results presented here establish the hinge region as an important element not only for LacI high-affinity operator binding but also for the essential communication between ligand binding domains. Moreover, the results confirm that DNA sequence/conformation can profoundly influence allostery for this prototypic regulatory protein.     

Modification of tyrosine residues of the lactose repressor protein.

Reaction of the lactose repressor protein from Escherichia coli with high molar excesses (up to 800 fold) of tetranitromethane resulted in modification of tyrosine residues in the amino-terminal and core regions of the molecule. Tyrosines 7 and 17 exhibit significant reactivity at low levels (5-10 fold molar excess) of tetranitromethane. The loss of operator binding activity upon nitration at these low concentrations of reagent indicates involvement of these two tyrosines in the binding process. Inducer binding activity was maintained at approx. 90% of unreacted repressor for all excesses of reagent studied. Addition of inducer to the repressor prior to reaction resulted in decreased modification of tyrosines in the core region, but anti-inducers did not affect the reaction significantly. The effect of inducers on the pattern of reaction apparently reflects the conformational change which occurs upon binding of these ligands. Acetylation of the repressor protein with N-acetylimidazole modified lysines and tyrosines with complete loss of operator binding activity and retention of 75-80% of inducer binding activity.     

Role of the purine repressor hinge sequence in repressor function.

A protease-hypersensitive hinge sequence in Escherichia coli purine repressor (PurR) connects an N-terminal DNA-binding domain with a contiguous corepressor-binding domain. Binding of one molecule of dimeric repressor to operator DNA protects the hinge against proteolytic cleavage. Mutations in the hinge region impair repressor function in vivo. Several nonfunctional hinge mutants were defective in low-affinity binding to operator DNA in the absence of corepressor as well as in high-affinity corepressor-dependent binding to operator DNA, although binding of corepressor was similar to binding of the wild-type repressor. These results establish a role for the hinge region in operator binding and lead to a proposal for two routes to form the holoPurR-operator complex.     

Thermodynamic analysis of unfolding and dissociation in lactose repressor protein.

Lactose repressor protein, regulator of lac enzyme expression in Escherichia coli, maintains its structure and function at extremely low protein concentrations (<10(-)12 M). To examine the unfolding and dissociation of this tetrameric protein, structural transitions in the presence of varying concentrations of urea were monitored by fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation, and functional activities. The spectroscopic data demonstrated a single cooperative transition with no evidence of folded dimeric or monomeric species of this protein. These spectroscopic transitions were reversible provided a long incubation step was employed in the refolding reaction at approximately 3 M urea. The refolded repressor protein possessed the same functional and structural properties as wild-type repressor protein. The absence of concentration dependence expected for tetramer dissociation to unfolded monomer (M4 <--> 4U) in the spectral transitions indicates that the disruption of the monomer-monomer interface and monomer unfolding are a concerted reaction (M4 <--> U4) that may occur prior to the dissociation of the dimer-dimer interface. Thus, we propose that the unfolded monomers remain associated at the C-terminus by the 4-helical coiled-coil structure that forms the dimer-dimer interface and that this intermediate is the end point detected in the spectral transitions. Efforts to confirm the existence of this species by ultracentrifugation were inhibited by the aggregation of this intermediate. Based upon these observations, the wild-type fluorescence and CD data were fit to a model, M4 <--> U4, which resulted in an overall DeltaG degrees for unfolding of 40 kcal/mol. Using a mutant protein, K84L, in which the monomer-monomer interface is stabilized, sedimentation equilibrium results demonstrated that the dimer-dimer interface of lac repressor could persist at higher levels of urea than the monomer-monomer interface. The tetramer-dimer transition monitored using this mutant repressor yields a DeltaG degrees of 20.4 kcal/mol. Using this free energy value for the dissociation process of U4 <--> 4U, an overall free energy change of approximately 60 kcal/mol was calculated for dissociation of all interfaces and unfolding of the tetrameric lac repressor, reflecting the exceptional stability of this protein.     

The lac repressor hinge helix in context: The effect of the DNA binding domain and symmetry

The Lac system of genes has been an important model system in understanding gene regulation. When the dimer lac repressor protein binds to the correct DNA sequence, the hinge region of the protein goes through a disorder to order transition. The hinge region is disordered when binding to nonoperator sequences. This region of the protein must pay a conformational entropic penalty to order when it is bound to operator DNA. Structural studies show that this region is flexible. Previous simulations showed that this region is disordered when free in solution without the DNA binding domain present. Our simulations corroborate that this region is extremely flexible in solution, but we find that the presence of the DNA binding domain proximal to the hinge helix and salt make the ordered conformation more favorable even without DNA present.     

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.     

Differential binding of allolactose anomers to the lactose repressor of Escherichia coli.

Preferential binding of the β-anomer of allolactose to the lactose repressor of Escherichia coli was demonstrated by two methods: (1) by repeated washing of ammonium sulfate precipitates of the allolactose-repressor complex and (2) by competitive inhibition of allolactose binding by isopropyl-β-d-thiogalacto-side. Quantitation showed that one β-allolactose binds per isopropyl-β-d-thiogalactoside binding site. A control system is postulated.     

DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro.

P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.     

Identification of functionally important residues in the DNA binding region of the mnt repressor

Single amino acid substitutions have been introduced throughout the N-terminal DNA binding region of the Mnt repressor, and the operator binding properties of the resulting mutant repressors have been assayed. These studies show that the side chains of Arg2, His6, Asn8, and Arg10 are critical for high affinity binding to operator DNA. Other side chains in the N-terminal region do not appear to play major roles in DNA recognition and binding. Specific alterations in the pattern of methylation protection afforded by the Arg2----Lys mutant protein suggest that Arg2 contacts the N7 groups of guanines 10 and 12 in the operator. In conjunction with previous results, these findings suggest that part of the N-terminal region of Mnt binds as an extended polypeptide strand within the major groove of the mnt operator.     

Trinitrobenzenesulfonate modification of the lysine residues in lactose repressor protein

Modification of the lysine residues in the lactose repressor protein has been carried out with trinitrobenzenesulfonate. Reaction of lysine residues at positions 33, 37, 108, 290, and 327 was observed. Inducer binding was increased by modification with this reagent, while both nonspecific DNA binding and operator DNA binding were diminished, although to differing degrees. The loss in operator DNA binding capacity was complete with modification of approximately 2 equiv of lysine per monomer. The extent of reaction was affected by the presence of both sugar and DNA ligands; binding activities of the modified protein and reaction pattern of the lysines were perturbed by these ligands. The presence of operator or nonspecific DNA during the reaction protected against specific and nonspecific DNA binding activity loss. This protection presumably occurs by steric restriction of reagent access to lysine residues which are essential for both nonspecific and operator binding interactions. Lysines-33 and -108 were protected from modification in the presence of DNA. These experiments suggest that the charge on the lysine residues is important for protein interaction with DNA and that steric constraints for operator DNA interaction with the protein are more restrictive than for nonspecific DNA binding. In contrast, inducer (isopropyl beta-D-thiogalactoside) presence partially protected lysine-290 from modification while significantly enhancing reaction at lysine-327. Conformational alterations consequent to inducer binding are apparently reflected in these altered lysine reactivities.