Importance of minor-groove contacts for recognition of DNA by the binding domain of Hin recombinase

Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein.     

Combinatorial redesign of the DNA binding specificity of a prokaryotic helix-turn-helix repressor

Redesign of the bacteriophage 434 Cro repressor was accomplished by using an in vivo genetic screening system to identify new variants that specifically bound previously unrecognized DNA sequences. Site-directed, combinatorial mutagenesis of the 434 Cro helix-turn-helix (HTH) motif generated libraries of new variants which were screened for binding to new target sequences. Multiple mutations of 434 Cro that functionally converted wild-type (wt) 434 Cro DNA binding-sequence specificity to that of a lambda bacteriophage-specific repressor were identified. The libraries contained variations within the HTH sequence at only three positions. In vivo and in vitro analysis of several of the identified 434 Cro variants showed that the relatively few changes in the recognition helix of the HTH motif of 434 Cro resulted in specific and tight binding of the target DNA sequences. For the best 434 Cro variant identified, an apparent K(d) for lambda O(R)3 of 1 nM was observed. In competition experiments, this Cro variant was observed to be highly selective. We conclude that functional 434 Cro repressor variants with new DNA binding specificities can be generated from wt 434 Cro by mutating just the recognition helix. Important characteristics of the screening system responsible for the successful identifications are discussed. Application of the techniques presented here may allow the identification of DNA binding protein variants that functionally affect DNA regulatory sequences important in disease and industrial and biotechnological processes.     

The role of the minor groove substituents in indirect readout of DNA sequence by 434 repressor

The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89-91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH(2) group on the purine bases at the binding site center. The N2-NH(2) group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH(2) group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface.     

Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs.

Single-chain derivatives of the phage 434 repressor, termed single-chain repressors, contain covalently dimerized DNA-binding domains (DBD) which are connected with a peptide linker in a head-to-tail arrangement. The prototype RR69 contains two wild-type DBDs, while RR*69 contains a wild-type and an engineered DBD. In this latter domain, the DNA- contacting amino acids of thealpha3 helix of the 434 repressor are replaced by the corresponding residues of the related P22 repressor. We have used binding site selection, targeted mutagenesis and binding affinity studies to define the optimum DNA recognition sequence for these single-chain proteins. It is shown that RR69 recognizes DNA sequences containing the consensus boxes of the 434 operators in a palindromic arrangement, and that RR*69 optimally binds to non-palindromic sequences containing a 434 operator box and a TTAA box of which the latter is present in most P22 operators. The spacing of these boxes, as in the 434 operators, is 6 bp. The DNA-binding of both single-chain repressors, similar to that of the 434 repressor, is influenced indirectly by the sequence of the non-contacted, spacer region. Thus, high affinity binding is dependent on both direct and indirect recognition. Nonetheless, the single-chain framework can accommodate certain substitutions to obtain altered DNA-binding specificity and RR*69 represents an example for the combination of altered direct and unchanged indirect readout mechanisms.     

Effect of salt shock on stability of lambdaimm434 lysogens

The affinities of the bacteriophage 434 repressor for its various binding sites depend on the type and/or concentration of monovalent cations. The ability of bacteriophage 434 repressor to govern the lysis-lysogeny decision depends on the DNA binding activities of the phage's cI repressor protein. We wished to determine whether changes in the intracellular ionic environment influence the lysis-lysogeny decision of the bacteriophage lambda(imm434). Our findings show that the ionic composition within bacterial cells varies with the cation concentration in the growth media. When lambda(imm434) lysogens were grown to mid-log or stationary phase and subsequently incubated in media with increasing monovalent salt concentrations, we observed a salt concentration-dependent increase in the frequency of bacteriophage spontaneous induction. We also found that the frequency of spontaneous induction varied with the type of monovalent cation in the medium. The salt-dependent increase in phage production was unaffected by a recA mutation. These findings indicate that the salt-dependent increase in phage production is not caused by activation of the SOS pathway. Instead, our evidence suggests that salt stress induces this lysogenic bacteriophage by interfering with 434 repressor-DNA interactions. We speculate that the salt-dependent increase in spontaneous induction is due to a direct effect on the repressor's affinity for DNA. Regardless of the precise mechanism, our findings demonstrate that salt stress can regulate the phage lysis-lysogeny switch.     

DNA recognition by the helix-turn-helix motif: investigation by laser Raman spectroscopy of the phage lambda repressor and its interaction with operator sites OL1 and OR3.

The lambda repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif. We describe laser Raman studies of the lambda operator sites OL1 and OR3 and their interaction with the DNA-binding domain of lambda repressor (residues 1-102). Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding. Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups. These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family. These perturbations differ between aqueous OL1 and OR3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation. The overall structure of the lambda domain is not greatly perturbed by binding to either OL1 or OR3, in accord with X-ray studies of other complexes. However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 in solution. This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions.     

Orientation of the Lac repressor DNA binding domain in complex with the left lac operator 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.     

Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor

It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition. We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate. We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor*. The specific DNA contacts made by repressor* are like those of 434 cro protein.     

Folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 Cro protein.

The phage 434 Cro protein, the N-terminal domain of its repressor (R1-69) and that of phage lambda (lambda6-85) constitute a group of small, monomeric, single-domain folding units consisting of five helices with striking structural similarity. The intrinsic helix stabilities in lambda6-85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in R1-69 in 7 M urea has been proposed as a folding initiation site. To understand the early events in the folding of 434 Cro, and for comparison with R1-69 and lambda6-85, we examined the conformational behavior of five peptides covering the entire 434 Cro sequence in water, 40% (by volume) TFE/water, and 7 M urea solutions using CD and NMR. Each peptide corresponds to a helix and adjacent residues as identified in the native 434 Cro NMR and crystal structures. All are soluble and monomeric in the solution conditions examined except for the peptide corresponding to the 434 Cro helix 4, which has low water solubility. Helix formation is observed for the 434 Cro helix 1 and helix 2 peptides in water, for all the peptides in 40% TFE and for none in 7 M urea. NMR data indicate that the helix limits in the peptides are similar to those in the native protein helices. The number of side-chain NOEs in water and TFE correlates with the helix content, and essentially none are observed in 7 M urea for any peptide, except that for helix 5, where a hydrophobic cluster may be present. The low intrinsic folding propensities of the five helices could account for the observed stability and folding behavior of 434 Cro and is, at least qualitatively, in accord with the results of the recently described diffusion-collision model incorporating intrinsic helix propensities.     

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12 mol/L-1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1×10-9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recog-nition properties previously determined for the RTRES domain. These operators containing the con-sensus sequence GTAAGAAARNTTACN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA inter-actions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.     

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.     

Recognition of DNA structure by 434 repressor.

In complexes of bacteriophage 434 binding sites with 434 repressor the central 4 bp of the 14 bp site are not contacted by the protein, although changes in these bases alter binding site affinity for the repressor. Our previous data suggested that the ability of the non-contacted central bases to be overtwisted in repressor-DNA complexes governs affinity of the binding site for 434 repressor. This idea was tested by examining the affinity of two central sequence variant 434 binding sites for 434 repressor as a function of binding site average twist. The 434 repressor preferred the relatively overwound binding site to the two more underwound forms. The greatest affinity enhancement resulting from increasing twist was observed with a binding site that is relatively underwound and more resistant to twisting deformation. Consistent with the idea that 434 repressor overtwists its binding site upon DNA binding, we show that 434 repressor is capable of binding to sites bearing a single base insertion in their center (a 15mer), but binds poorly to binding sites bearing central base deletions (12mer and 13mer). The N-terminal dimer interface plays a large role in determining 434 repressor central base preferences. Mutations in this interface eliminate central base discrimination and/or site size preferences. These mutations also lead to changes in the size of the repressor footprint on the various sized DNA sites that are consistent with their binding characteristics.     

Recognition of DNA sequences by the repressor of bacteriophage 434

The structure of a complex between the DNA-binding domain of phage 434 repressor and a 14 base-pair synthetic DNA operator reveals the molecular interactions important for sequence-specific recognition. A set of contacts with DNA backbone, notably involving hydrogen bonds between peptide-NH groups and DNA phosphates, position the repressor and fix the DNA configuration. Direct interactions between amino acid side chains and DNA bases involve nonpolar van der Waals contacts as well as hydrogen bonds. The structures of the repressor domain and of the 434 cro protein are extremely similar. There appear to be no major conformational changes in the proteins when they bind to DNA.     

Comparison of operator-specific and nonspecific DNA binding of the lambda cI repressor: [KCl] and pH effects.

The effects of proton and KCl activity on the nonspecific lambda cI repressor-DNA interactions and on the site-specific repressor-O(R) interactions were compared, in order to assess their roles in site specificity. The repressor-O(R) interactions were studied by using DNase I footprint titration. The Gibbs free energy changes for binding and for cooperativity were determined between 25 and 300 mM KCl, from individual-site isotherms for the binding of repressor to O(R) and to reduced-valency mutants. The proton-linked effects on repressor-O(R) interactions have been published [Senear, D. F., & Ackers, G. K. (1990) Biochemistry 29, 6568-6577; Senear, D. F., & Bolen, D. W. (1991) Methods Enzymol. (in press)]. Nonspecific binding was studied by using a nitrocellulose filter binding assay, which proved advantageous in this case, due to the relatively weak nonspecific binding, and precipitation of repressor-DNA complexes. Filter binding provided measurements at low binding density where precipitation did not occur. The data provide estimates of the Gibbs free energy changes for nonspecific, intrinsic binding, but not for cooperativity. The KCl concentration dependencies of the intrinsic binding constants indicate that ion release plays similar roles in distinguishing between the operators and in discriminating operator from nonoperator DNA. Binding to DNA is accompanied by net proton absorption. Near neutral pH, proton linkages to operator and nonoperator binding are the same. Differences at acid and at basic pH implicate the same ionizable repressor groups in distinguishing between the operators and in discriminating operator from nonoperator DNA. The results indicate similar overall modes of operator and nonoperator binding of repressor, but implicate indirect effects of DNA sequence as important contributors to sequence recognition.