Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turn-helix (HTH) motif of the Trp repressor, and guanine in position 9 of the α-centred consensustrp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical α-or β-centredtrp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the α-centredtrp operator. There are also interactions with other bases in positions 8 and 9 of the α-centredtrp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus α-centredtrp operator and a similartrp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the β-centredtrp operator shows a change in the specificity of binding to a β-centred symmetricaltrp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the α-centredtrp operator.     

The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding

We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix. We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator. We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix. Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator. They further suggest that residue 9 of the recognition helix (asparagine 25) interacts non-specifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3.     

Isolation and analysis of arc repressor mutants: evidence for an unusual mechanism of DNA binding

We have isolated 64 different missense mutations at 36 out of 53 residue positions in the Arc repressor of bacteriophage P22. Many of the mutant proteins with substitutions in the C-terminal 40 residues of Arc have reduced intracellular levels and probably have altered structures or stabilities. Mutations in the N-terminal ten residues of Arc cause large decreases in operator DNA binding affinity without affecting the ability of Arc to fold into a stable three-dimensional structure. We argue that these N-terminal residues are important for operator recognition but that they are not part of a conventional helix-turn-helix DNA binding structure. These results suggest that Arc may use a new mechanism for sequence specific DNA binding.     

Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turn-helix (HTH) motif of the Trp repressor, and guanine in position 9 of the -centred consensustrp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical -or -centredtrp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the -centredtrp operator. There are also interactions with other bases in positions 8 and 9 of the -centredtrp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus -centredtrp operator and a similartrp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the -centredtrp operator shows a change in the specificity of binding to a -centred symmetricaltrp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the -centredtrp operator.     

Sequence-specific 1H NMR assignment and secondary structure of the Arc repressor of bacteriophage P22, as determined by two-dimensional 1H NMR spectroscopy

The Arc repressor of bacteriophage P22 is a DNA binding protein that does not belong to any of the known classes of such proteins. We have undertaken a 1H NMR study of the protein with the aim of elucidating its three-dimensional structure in solution and its mode of binding of operator DNA. Here we present the 1H nuclear magnetic resonance (NMR) assignments of all backbone protons and most of the side-chain protons of Arc repressor. Elements of secondary structure have been identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser enhancements (NOEs). Two alpha-helical regions have been found in the peptide regions 16-29 and 35-45. The ends of the helices could not yet be firmly established and could extend to residue 31 for the first helix and to residue 49 for the second. Immediately before the first helix, between residues 8 and 14, a region is present with beta-sheet characteristics dominated by a close proximity of the alpha-protons of residues 9 and 13. Because of the dimeric nature of the protein there are still two possible ways in which the NOEs in the beta-sheet region can be interpreted. If the NOEs are intramonomer, this requires a tight turn involving residues 10-12. Alternatively, if the NOEs are intermonomer, then and antiparallel beta-sheet would be implicated comprising two strands of different Arc monomers. While the data presently do not allow an unambiguous choice between these two possibilities, some evidence is discussed that favors the latter (beta-sheet between monomers).(ABSTRACT TRUNCATED AT 250 WORDS)     

lac repressor mutants with double or triple exchanges in the recognition helix bind specifically to lac operator variants with multiple exchanges.

Several lac repressor mutants have been isolated which repress beta-galactosidase synthesis in Escherichia coli up to 200-fold. They do so by binding specifically to particular symmetrical lac Oc operator variants. The mutations in the lac repressor are localized in two separate parts of the recognition helix comprising (i) residues 1 and 2 which interact with base pairs 4 and 5 of lac operator and (ii) residue 6 which recognizes operator base pair 6. Mutations of residues 1 and 2 may be combined with a mutation of residue 6. The resulting mutant protein binds specifically to an operator variant with three symmetric exchanges in base pairs 4, 5 and 6.     

Specificity of Mnt 'master residue' obtained from in vivo and in vitro selections

Mnt is a repressor from phage P22 that belongs to the ribbon–helix–helix family of DNA binding factors. Four amino acids from the N-terminus of the protein, Arg2, His6, Asn8 and Arg10, interact with the base pairs of the DNA to provide the sequence specificity. Raumann et al. (Nature Struct. Biol., 2, 1115–1122) identified position 6 as a ‘master residue’ that controls the specificity of the protein. Models for the interaction have residue 6 of Mnt interacting directly with position 5 of the operator. In vivo selections demonstrated that protein variants at residue 6 bound specifically to operator mutations at that position. Operators in which the wild-type G at position 5 was replaced by T specifically bound to several different protein variants, primarily hydrophobic residues. The obtained protein variants, plus some others, were used in in vitro selections to determine their preferred binding sites. The results showed that the residue at position 6 influenced the preference for binding site bases predominantly at position 5, but that the effects of altering it can extend over longer distances, consistent with its designation as a ‘master residue’. The similarities of binding sites for different residues do not correlate strongly with common measures of amino acid similarities.     

Interaction of the bacteriophage P22 Arc repressor with operator DNA

Are repressor binds to a single, partially symmetric, 21 base-pair operator site that is centered between the -10 and -35 regions of the Pant promoter. Protection and interference experiments show that Arc makes contacts with the operator on one side of the DNA helix. Although Arc is a small protein (53 residues/subunit), it makes contacts that are farther from the center of the operator than those made by many larger repressors. These extended contacts include the phosphate groups at the ends of the 21 base-pair site. Under standard conditions (pH 7.5, 100 mM-KCl, 3 mM-MgCl2, 22 degrees C) half-maximal operator binding is observed at an Arc concentration of 2.5 X 10(-9) M and the protein-DNA complex is very stable (t1/2 approximately equal to 80 min).     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of β-galactosidase with symmetric variants of α- and β-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the α-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the α-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for α-centered trp operator variants with exchanges in positions 3, 4 and 5.     

Isolation and characterization of LexA mutant repressors with enhanced DNA binding affinity.

The LexA repressor from Escherichia coli is a sequence-specific DNA binding protein that shows no pronounced sequence homology with any of the known structural motifs involved in DNA binding. Since little is known about how this protein interacts with DNA, we have selected and characterized a great number of intragenic, second-site mutations which restored at least partially the activity of LexA mutant repressors deficient in DNA binding. In 47 cases, the suppressor effect of these mutations was due to an Ind- phenotype leading presumably to a stabilization of the mutant protein. With one exception, these second-site mutations are all found in a small cluster (amino acid residues 80 to 85) including the LexA cleavage site between amino acid residues 84 and 85 and include both already known Ind- mutations as well as new variants like GN80, GS80, VL82 and AV84. The remaining 26 independently isolated second-site suppressor mutations all mapped within the amino-terminal DNA binding domain of LexA, at positions 22 (situated in the turn between helix 1 and helix 2) and positions 57, 59, 62, 71 and 73. These latter amino acid residues are all found beyond helix 3, in a region where we have previously identified a cluster of LexA (Def) mutant repressors. In several cases the parental LexA (Def) mutation has been removed by subcloning or site-directed mutagenesis. With one exception, these LexA variants show tighter in vivo repression than the LexA wild-type repressor. The most strongly improved variant (LexA EK71, i.e. Glu71----Lys) that shows an about threefold increased repression rate in vivo, was purified and its binding to a short consensus operator DNA fragment studied using a modified nitrocellulose filter binding assay. As expected from the in vivo data, LexA EK71 interacts more tightly with both operator and (more dramatically) with non-operator DNA. A determination of the equilibrium association constants of LexA EK71 and LexA wild-type as a function of monovalent salt concentration suggests that LexA EK71 might form an additional ionic interaction with operator DNA as compared to the LexA wild-type repressor. A comparison of the binding of LexA to a non-operator DNA fragment further shows that LexA interacts with the consensus operator very selectively with a specificity factor of Ks/Kns of 1.4 x 10(6) under near-physiological salt conditions.     

Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions

Hydrophobic substitutions at solvent-exposed positions in two alpha-helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild-type Arc, with a rough correlation between the number of polar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a t(m) 6 degrees C higher than wild-type. By contrast, a helix A mutant with three similar polar-to-hydrophobic substitutions was 23 degrees C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in helix B was tolerated when introduced into the wild-type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues.     

Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.

The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.     

Degradation of the DNA-binding domain of wild-type and i-d lac repressors in Escherichia coli.

It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i-d repressors), could be divided into two groups by their capacity or incapacity to bind non-specifically to the phosphate groups of the DNA backbone. All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal of residue 52 showed uneffected non-specific DNA binding. All 13 tested i-d repressors with amino acid substitutions to the N-terminal of residue 53 did not bind to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded. The degradation in vivo only affects the amino-terminal 50-60 residues producing a mutant-specific pattern of stable repressor fragments. These fragments are tetrameric and capable of binding inducer in vivo and in vitro. The proteolytic attack presumably takes place during synthesis of the i-d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse-chase experiment) and in vitro. The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media. Wild-type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media. The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i-d lac repressor proteins affect the three-dimensional structure of the amino-terminal DNA-binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases.     

Role of residue 147 in the gene regulatory function of the Escherichia coli purine repressor.

The crystal structures of corepressor-bound and free Escherichia coli purine repressor (PurR) have delineated the roles of several residues in corepressor binding and specificity and the intramolecular signal transduction (allosterism) of this LacI/GalR family member. From these structures, residue W147 was implicated as a key component of the allosteric response, but in many members of the LacI/GalR family, position 147 is occupied by an arginine. To understand the role of this tryptophan at position 147, three proteins, substituted by phenylalanine (W147F), alanine (W147A), or arginine (W147R), were constructed and characterized in vivo and in vitro, and their structures were determined. W147F displays a decreased affinity for corepressor and is a poor repressor in vivo. W147A and W147R, on the other hand, are super repressors and bind corepressor 13.6 and 7.9 times more tightly, respectively, than wild-type. Each mutant PurR-hypoxanthine-purF operator holo complex crystallizes isomorphously to wild-type. Whereas the apo corepressor binding domain (CBD) of W147F crystallizes under those conditions used for the wild-type protein, neither the apo CBD of W147R nor W147A crystallizes, although screened extensively for new crystal forms. Structures of the holo repressor mutants have been solved to resolutions between 2.5 and 2.9 A, and the structure of the apo CBD of W147F has been solved to 2.4 A resolution. These structures provide insight into the altered biochemical properties and physiological functions of these mutants, which appear to depend on the sometimes subtle preference for one conformation (apo vs holo) over the other.     

Increased sequence hydrophobicity reduces conformational specificity: A mutational case study of the Arc repressor protein

The amino-acid sequences of soluble, globular proteins must have hydrophobic residues to form a stable core, but excess sequence hydrophobicity can lead to loss of native state conformational specificity and aggregation. Previous studies of polar-to-hydrophobic mutations in the β-sheet of the Arc repressor dimer showed that a single substitution at position 11 (N11L) leads to population of an alternate dimeric fold in which the β-sheet is replaced by helix. Two additional hydrophobic mutations at positions 9 and 13 (Q9V and R13V) lead to population of a differently folded octamer along with both dimeric folds. Here we conduct a comprehensive study of the sequence determinants of this progressive loss of fold specificity. We find that the alternate dimer-fold specifically results from the N11L substitution and is not promoted by other hydrophobic substitutions in the β-sheet. We also find that three highly hydrophobic substitutions at positions 9, 11, and 13 are necessary and sufficient for oligomer formation, but the oligomer size depends on the identity of the hydrophobic residue in question. The hydrophobic substitutions increase thermal stability, illustrating how increased hydrophobicity can increase folding stability even as it degrades conformational specificity. The oligomeric variants are predicted to be aggregation-prone but may be hindered from doing so by proline residues that flank the β-sheet region. Loss of conformational specificity due to increased hydrophobicity can manifest itself at any level of structure, depending upon the specific mutations and the context in which they occur.