An alkaline phosphatase protection assay to investigate trp repressor/operator interactions

We have used an alkaline phosphatase protection assay to investigate the interaction of the trp repressor with its operator sequence. The assay is based on the principle that the trp repressor will protect a terminally 5'-32P-labeled operator DNA fragment from attack by alkaline phosphatase. The optimal oligonucleotide for investigating the trp repressor/operator interaction extends two base pairs from each end of the genetically defined target sequence predicted by in vivo studies [Bass et al. (1987) Genes Dev. 1, 565-572]. The assay works well over a 10,000-fold range of protein/DNA affinity and is used to show that the corepressor, L-tryptophan, causes the liganded repressor to bind a 20 base pair trp operator duplex 6400 times more strongly than the unliganded aporepressor. The affinity of the trp repressor for operators containing symmetrical mutations was interpreted in terms of the trp repressor/operator crystal structure as follows: (1) Direct hydrogen bonds with the functional groups of G-9 of the trp operator and the side chain of Arg 69 of the trp repressor contribute to DNA-binding specificity. (2) G-6 of the trp operator is critical for DNA-binding specificity probably because of the two water-mediated hydrogen bonds between its functional groups and the N-terminus of the trp repressor's E-helix. (3) Sequence-dependent aspects of the trp operator's conformation help stabilize the trp repressor/operator complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flexibility of the DNA-binding domains of trp repressor

An orthorhombic crystal form of trp repressor (aporepressor plus L-tryptophan ligand) was solved by molecular replacement, refined to 1.65 A resolution, and compared to the structure of the repressor in trigonal crystals. Even though these two crystal forms of repressor were grown under identical conditions, the refined structures have distinctly different conformations of the DNA-binding domains. Unlike the repressor/aporepressor structural transition, the conformational shift is not caused by the binding or loss of the L-tryptophan ligand. We conclude that while L-tryptophan binding is essential for forming a specific complex with trp operator DNA, the corepressor ligand does not lock the repressor into a single conformation that is complementary to the operator. This flexibility may be required by the various binding modes proposed for trp repressor in its search for and adherence to its three different operator sites.     

Site-specific intercalation at the triplex-duplex junction induces a conformational change which is detectable by hypersensitivity to diethylpyrocarbonate.

Using site-specific intercalation directed by intermolecular triplex formation, the conformation of an intercalation site in DNA was examined by footprinting with the purine-specific (A much greater than G) reagent diethylpyrocarbonate. Site specific intercalation was achieved by covalently linking an intercalator to the 5' end of a homopyrimidine oligodeoxynucleotide, which bound to a homopurinehomopyrimidine stretch in a recombinant plasmid via intermolecular triplex formation. This directs intercalation to a single site in 3kb of DNA at the 5' triplex-duplex junction. Footprinting with diethylpyrocarbonate and dimethylsulphate revealed strong protection from modification of adenine residues within the triple-helix in concordance with their Hoogsteen pairing with the third strand, and a strong hypersensitivity to diethylpyrocarbonate at the first adenine of the duplex. This result indicates that intercalation at this site induces a conformational change at the 5' triplex-duplex junction. Furthermore, the same diethlypyrocarbonate hypersensitivity was observed with an unmodified triple-strand forming oligonucleotide and a range of intercalating molecules present in solution. Thus the 5' triplex-duplex junction is a strong binding site for some intercalating molecules and the junction undergoes a conformational change which is sensitive to diethylpyrocarbonate upon insertion of the planar aromatic chromophore. This conformational change can be used to direct a single-strand cut in duplex DNA to a defined site.     

Stereochemical effects of L-tryptophan and its analogues on trp repressor's affinity for operator-DNA

We have employed a filter binding assay to help study the mechanism by which bound L-tryptophan enables the Escherichia coli trp repressor to bind its operators. We have prepared variants of the trp repressor using structural analogues of the natural corepressor, L-tryptophan, and measured the affinity of these variants for a 20-base pair oligonucleotide duplex containing a symmetrical idealization of the trp operator from the E. coli trpEDCBA operon. By normalizing for each analogue's previously determined affinity for the trp aporepressor, we have estimated the extent to which each of the functional groups of bound L-tryptophan contributes to operator affinity. We discuss the likely role of these functional groups in the context of the crystal structures of the inactive, unliganded trp aporepressor, the liganded, active repressor, an inactive pseudorepressor (Pseudorepressors are formed by analogues of L-tryptophan that bind at the tryptophan-binding site but form near isomorphs of the repressor that have poor affinity for operator-DNA.) and the trp repressor/operator complex. We find that the alpha-amino group and an unsubstituted amino (-NH-) nitrogen of L-tryptophan's indole ring are essential for operator affinity. The former properly orients the corepressor and the latter interacts directly with the DNA. The alpha-carboxyl group, on the other hand, greatly enhances but is not essential for operator binding. The alpha-carboxylate's role, which is dependent on the corepressor's orientation in the binding pocket, is apparently to position the guanidinium group of Arg-84 for favorable contacts with the operator's sugar-phosphate backbone.     

The thermodynamics of polyamide-DNA recognition: hairpin polyamide binding in the minor groove of duplex DNA

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.     

The fifth Datta Lecture. Structural similarities between the aspartate receptor of bacterial chemotaxis and the trp repressor of E. coli. Implications for transmembrane signaling.

A high resolution structure of the N-terminal ligand-binding domain of the aspartate receptor which mediates aspartate chemotaxis in Salmonella typhimurium has recently been reported. A least-squares superposition of the alpha-amino nitrogen, alpha-carbon, beta-carbon, and alpha-carboxylate carbon of the aspartate bound to the aspartate receptor onto the equivalent atoms in the tryptophan bound to the trp repressor provides evidence for similarity between key parts of the active sites that bind to the alpha-amino and alpha-carboxylates of the respective ligands. Because the N-terminal domain of the aspartate receptor and the trp repressor also share other structural similarities, we hypothesize that the similarity between the aspartate receptor and the trp repressor derives from a similarity in ligand-induced conformational changes at the active sites of these proteins. This hypothesis also implies that an important signaling event in the aspartate receptor occurs through tertiary conformational changes within a single subunit.     

DNA-induced conformational changes in bacteriophage 434 repressor

Although bacteriophage 434 repressor binds to its specific DNA sites only as a dimer, formation of the dimers in solution occurs at concentrations three orders of magnitude higher than those needed to bind the 434 operator DNA. Our results suggest that both specific and non-specific DNA induce conformational changes in repressor that lead to formation of repressor dimers. The repressor conformational changes induced by DNA occur at concentrations much lower than those needed for binding of repressor, suggesting that the alternative conformations of repressor persist even if the protein is not in direct contact with DNA. Hence, DNA acts in a "catalytic" fashion to induce a steady-state amount of an alternative repressor conformation that has an enhanced affinity for its specific binding site. These findings suggest that the repressor conformer induced by non-specific DNA is the form of the repressor that is optimized for searching for DNA binding sites along non-specific DNA. Upon finding a binding site, the repressor protein undergoes an additional conformational change that allows it to "lock-on" to its specific site.     

The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid

The binding of the corepressor, L-tryptophan, and an inducer, indole propanoic acid, to the trp repressor from Escherichia coli was studied by absorbance, fluorescence, circular dichroic and proton NMR spectroscopy. The two ligands bind to the same site on the repressor in the same orientation; they are molecular competitors. The binding site is of relatively low polarity and contains at least one methyl group that lies 0.3 nm over the indole moiety near the C5 proton of the bound ligand, and an aromatic residue, probably tyrosine. The dissociation constant was determined as a function of temperature and pH. At 25 degrees C in 0.1 M phosphate buffer, pH 7.6, the dissociation constant is 18 +/- 2 microM for both ligands. In the same buffer system, the van't Hoff enthalpy for dissociation is 35.5 +/- 1 kJ/mol for tryptophan, and 30.5 +/- 2 kJ/mol for indole propanoic acid. The affinity of the repressor for indole propanoic acid is independent of pH in the range 7 less than 10, but decreases four fold for tryptophan in the same range. The amino group of tryptophan makes a significant contribution to its binding affinity. Difference NMR spectra showed that there are few changes of protein resonances on binding ligands. The NMR signals of the bound resonances were assigned by difference and nuclear Overhauser effect spectroscopy. The properties of the bound resonances are consistent with the ligands being largely immobilised within the binding site. The difference spectra, and the known functional differences of the two ligands, suggest that tryptophan induces a slightly different conformational state in the repressor from that induced by indole propanoic acid. There is no evidence for a global transition. The rate of dissociation of ligands is relatively large, being in the range 400-600 s-1.     

Sequence recognition in the minor groove of DNA by covalently linked formamido imidazole-pyrrole-imidazole polyamides: effect of H-pin linkage and linker length on selectivity and affinity

The polyamide N-formamido imidazole-pyrrole-imidazole (f-ImPyIm) binds with an exceptionally high affinity for its cognate site 5'-ACGCGT-3' as a stacked, staggered, and noncovalent cooperative dimer. Investigations are presented into its sequence specificity and binding affinity when linked covalently as an H-pin "dimer". Five f-ImPyIm cross-linked analogues with six to nine methylene linkers and an eight-linked ethylene glycol linker were examined to investigate the effect of linkage and linker length on DNA binding. Thermal denaturation studies on short DNA hairpins showed preferential binding by both f-ImPyIm (DeltaTm = 7.8 degrees C) and its cross-linked derivatives (DeltaTm > 30 degrees C) at 5'-ACGCGT-3', indicating sequence specificity was retained on linkage. DNase I footprinting confirmed strict cognate site selectivity and demonstrated that affinity increased with linker length (f-ImPyIm-9 = f-ImPyIm-8 = f-ImPyIm-EG-8 > f-ImPyIm-7 > f-ImPyIm-6). The eight- and nine-linked derivatives bound at 100-fold lower concentrations at the cognate site relative to f-ImPyIm-6, and with 10-fold higher affinity than unlinked f-ImPyIm. Use of an ethylene glycol linkage in f-ImPyIm-EG-8 to improve solubility slightly increased the cognate site affinity relative to those of f-ImPyIm-8 and f-ImPyIm-9, although some selectivity was lost at high ligand concentration. CD demonstrated that cognate site binding by eight and nine-linked compounds occurred in the minor groove. SPR analysis gave a binding affinity (K) for f-ImPyIm-EG-8 at the cognate site of 2 x 10(10) M-1, representing a 100-fold increase relative to that of f-ImPyIm. This study demonstrates that the high-affinity cooperative binding of f-ImPyIm can be enhanced significantly by suitable covalent linkage, while maintaining its strict cognate site selectivity.     

Alternative heterocycles for DNA recognition: a 3-pyrazole/pyrrole pair specifies for G.C base pairs

Synthetic ligands comprising three aromatic amino acids, pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp), specifically recognize predetermined sequences as side-by-side pairs in the minor groove of DNA. To expand the repertoire of aromatic rings that may be utilized for minor groove recognition, three five-membered heterocyclic rings, 3-pyrazolecarboxylic acid (3-Pz), 4-pyrazolecarboxylic acid (4-Pz), and furan-2-carboxylic acid (Fr), were examined at the N-terminus of eight-ring hairpin polyamide ligands. The DNA binding properties of 3-Pz, 4-Pz, and Fr each paired with Py were studied by quantitative DNase I footprinting titrations on a 283 bp DNA restriction fragment containing four 6-bp binding sites 5'-ATNCCTAA-3' (N = G, C, A, or T; 6-bp polyamide binding site is underlined). The pair 3-Pz/Py has increased binding affinity and sequence specificity for G.C bp compared with Im/Py.     

Novel diamino imidazole and pyrrole-containing polyamides: Synthesis and DNA binding studies of mono- and diamino-phenyl-ImPy*Im polyamides designed to target 5'-ACGCGT-3'

Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5'-ACGCGT-3'. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔT(M), DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPyIm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5'-ACGCGT-3'. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPyIm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 × 10(7)M(-1), which is ～3 times higher than that for its monoamino analog 3 (4.8 × 10(6)M(-1)). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5 over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2.     

A negative electrostatic determinant mediates the association between the Escherichia coli trp repressor and its operator DNA.

The electrostatic potential surfaces were characterized for trp repressor models that bind to DNA with sequence specificity, without specificity, and not at all. Comparisons among the surfaces were used to isolate protein surface features likely to be important in DNA binding. Models that differ in protein conformation and tryptophan-analogue binding consistently showed positive potential associated with the protein surfaces that interact with the DNA major groove. However, negative potential is associated with the trp repressor surface that contacts the DNA minor groove. This negative potential is significantly neutralized in the protein conformation that is bound to DNA. Positive potential is also associated with the tryptophan binding-site surface, a consequence of the tryptophan- or tryptophan analogue-induced allosteric change. This protein region is complementary to the strongest negative potential associated with the DNA phosphate backbone and is also present in the isolated protein structure from the protein-DNA complex. The effects of charge-change mutation, pH dependence, and salt dependence on the electrostatic potential surfaces were also examined with regard to their effects on protein-DNA binding constants. A consistent model is formed that defines a role for long-range electrostatics early in the protein-DNA association process and complements previous structural, molecular association, and mutagenesis studies.     

Structural studies of E. coli topoisomerase III-DNA complexes reveal a novel type IA topoisomerase-DNA conformational intermediate

Escherichia coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5' phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an eight-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is bound along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding.     

Synthesis of nonlinear DNA-binding peptide with binding specificity determinants close to those of 434 Cro-repressor

Design, synthesis and DNA binding activity of a nonlinear 102 residue peptide are reported. The peptide contains four sequence-specific DNA binding domains of 434 Cro protein. These four domains were linked covalently to a symmetrical carboxyterminal crosslinker that contains four arms each ending with an aliphatic aminogroup. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha helical, beta-sheet and random coiled conformations with an alpha helical content of about 16% at room temperature. The alpha helicity is increased up to 40% in the presence of 40% trifluoroethanol. Upon complex formation between the peptide and DNA a change in the peptide conformation takes place which is consistent with an alpha-beta transition in the DNA binding, helix-turn-helix motif of 434 Cro repressor. Evidently residues present in helices alpha(2) and alpha(3) form a beta hairpin which is inserted in the minor DNA groove. The latter inference is supported by our observations that the peptide can displace minor groove binding antibiotic distamycin A from a complex with poly(dA).poly(dT). As revealed from DNase protection studies the peptide exhibits preferences for binding to operator and pseudooperator sites recognized by 434 Cro repressor. It binds strongly to operator sites OR1, OR2 and OR3 and exhibits a greater affinity for pseudooperator site Op1. From analysis of nucleotide sequences in the strong affinity binding sites for the peptide on DNA a conclusion is drawn that it binds to pseudosymmetrical nucleotide sequences 5'-ACAA(W)nCTGT-3', where W is an arbitrary nucleotide. n is equal to six or seven. In the strongest affinity binding site for the peptide on DNA (Op1) motif 5'-ACAA-3' is replaced by sequence 5'-ACCA-3'. A difference in binding specificity shown by the peptide and 434 Cro protein could be attributed to a flexibility of the connecting chains between DNA-binding domains in the peptide molecule as well as to a replacement of Thr - Ala in the alpha 2 helix. Removal of two residues from the N-terminal end of helix alpha 2 in each of the four DNA binding domains of 434 Cro present in the peptide leads to a loss of binding specificity, although the modified peptide binds to DNA unspecifically.     

Recognition of the DNA minor groove by pyrrole-imidazole polyamides: comparison of desmethyl- and N-methylpyrrole

Polyamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), and N-methyl-3-hydroxypyrrole (Hp) are synthetic ligands that recognize predetermined DNA sequences with affinities and specificities comparable to many DNA-binding proteins. As derivatives of the natural products distamycin and netropsin, Py/Im/Hp polyamides have retained the N-methyl substituent, although structural studies of polyamide:DNA complexes have not revealed an obvious function for the N-methyl. In order to assess the role of the N-methyl moiety in polyamide:DNA recognition, a new monomer, desmethylpyrrole (Ds), where the N-methyl moiety has been replaced with hydrogen, was incorporated into an eight-ring hairpin polyamide by solid-phase synthesis. MPE footprinting, affinity cleavage, and quantitative DNase I footprinting revealed that replacement of each Py residue with Ds resulted in identical binding site size and orientation and similar binding affinity for the six-base-pair (bp) target DNA sequence. Remarkably, the Ds-containing polyamide exhibited an 8-fold loss in specificity for the match site versus a mismatched DNA site, relative to the all-Py parent. Polyamides with Ds exhibit increased water solubility, which may alter the cell membrane permeability properties of the polyamide. The addition of Ds to the repertoire of available monomers may prove useful as polyamides are applied to gene regulation in vivo. However, the benefits of Ds incorporation must be balanced with a potential loss in specificity.