The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions.

The interaction of the dimeric Escherichia coli purine repressor (PurR) with its cognate sequences leads to a 45 degrees to 50 degrees kink at a central CpG base step towards the major groove, as dyad-related leucine side-chains interdigitate between these bases from the minor groove. The resulting broadening of the minor groove increases the accessibility of the six central base-pairs towards minor groove interactions with residues from PurR. It has been shown that lysine 55 of PurR makes a direct contact with the adenine base (Ade8) directly 5' to the central CpG base-pair step in the high-affinity purF operator sequence. We have investigated the importance of this interaction in the specificity and affinity of wild-type PurR (WT) for its operators and we have studied a mutant of PurR in which Lys55 is replaced with alanine (K55A). Complexes of WT and K55A with duplex DNA containing pur operator sequences varied at position 8 were investigated crystallographically, and binding studies were performed using fluorescence anisotropy. The structures of the protein-DNA complexes reveal a relatively unperturbed global conformation regardless of the identity of the base-pair at position 8 or residue 55. In all structures the combination of higher resolution and a palindromic purF operator site allowed several new PurR.DNA interactions to be observed, including contacts by Thr15, Thr16 and His20. The side-chain of Lys55 makes productive, though varying, interactions with the adenine, thymine or cytosine base at position 8 that result in equilibrium dissociation constants of 2.6 nM, 10 nM and 35 nM, respectively. However, the bulk of the lysine side-chain apparently blocks high-affinity binding of operators with guanine at position 8 (Kd620 nM). Also, the high-affinity binding conformation appears blocked, as crystals of WT bound to DNA with guanine at position 8 could not be grown. In complexes containing K55A, the alanine side-chain is too far removed to engage in van der Waals interactions with the operator, and, with the loss of the general electrostatic interaction between the phosphate backbone and the ammonium group of lysine, K55A binds each operator weakly. However, the mutation leads to a swap of specificity of PurR for the base at position 8, with K55A exhibiting a twofold preference for guanine over adenine. In addition to defining the role of Lys55 in PurR minor groove binding, these studies provide structural insight into the minor groove binding specificities of other LacI/GalR family members that have either alanine (e.g. LacI, GalR, CcpA) or a basic residue (e.g. RafR, ScrR, RbtR) at the comparable position.

Glasfeld A ，Koehler AN ，Schumacher MA ，Brennan RG ... -  《JOURNAL OF MOLECULAR BIOLOGY》

The arginine repressor of Escherichia coli K-12 makes direct contacts to minor and major groove determinants of the operators.

In order to gain further insight into the molecular mechanism of arginine-dependent operator recognition by the hexameric Escherichia coli arginine repressor we have probed protein-DNA interactions in vitro and in vivo. We have extensively applied the chemical modification-protection and premodification-interference approach to two operators, the natural operator overlapping the P2 promoter of the carAB operon and a fully symmetrical consensus sequence. Backbone contacts were revealed by hydroxyl radical footprinting and phosphate ethylation interference. Base-specific contacts to purines and pyrimidines were revealed by methylation protection and premodification interference, KMnO4 and NH2OH.HCl-specific modification of thymine and cytosine residues, base-removal (depurination and depyrimidation), and base substitution (uracil and inosine). Additional information on the groove specificity of repressor binding was obtained by small ligand binding interference (distamycin and methyl green). In vivo, we measured the effects on the repressibility of 24 single base-pair substitutions obtained by saturation mutagenesis of half an Arg box in the carAB operator. The results of these experiments point to the conclusion that a hexameric arginine repressor molecule covers four turns of the helix, makes base-specific contacts to at least one guanine (G4 or G4') and two thymine (T3, T13', or T3', T13) residues in each one of four consecutive major grooves on one face of the helix and with four A-T/T-A base-pairs, comprising the adenine residues A9, 9', 12, 12' and the thymine residues T10, 10', 11, 11', in the two outermost minor grooves of the operator, on the very same face of the DNA molecule. The hydrophobic 5-methyl groups of four thymine residues (T3, 3', 13, 13') in each Arg box contribute to major groove-specific recognition via hydrophobic and/or van der Waals interactions. The importance of minor groove contacts was further supported by the drastic effect of distamycin binding interference. In vivo, the most pronounced drops in repressibility were occasioned by mutations at positions 10 (A-->G or C), 11 (T-->A or G) and 12 (A-->G, T or C).

Wang H ，Glansdorff N ，Charlier D 《JOURNAL OF MOLECULAR BIOLOGY》

Transcription regulation in thermophilic bacteria: high resolution contact probing of Bacillus stearothermophilus and Thermotoga neapolitana arginine repressor-operator interactions.

Arginine-mediated regulation is remarkably well conserved in very divergent bacteria, and shows a number of unusual features that distinguish arginine regulation from other transcriptional control mechanisms. The arginine repressor subunit consists of a basic N-terminal DNA-binding domain, which belongs to the winged helix-turn-helix family, connected through a flexible linker to an acidic C-terminal domain responsible for binding of arginine and assembly of the high-affinity holohexamer, which binds an approximately 40 bp target. To gain further insight into the molecular details of arginine repressor-operator interactions we have established a high resolution contact map of the argC operator from Bacillus stearothermophilus, a moderate thermophilic Gram-positive bacterium, and the argR operator from Thermotoga neapolitana, a Gram-negative hyperthermophile, with the corresponding ArgR proteins. Enzymatic and chemical footprinting have been combined with missing contact, pre-modification, base substitution, and small ligand binding interference techniques to gather information on backbone and base-specific contacts with major and minor groove determinants of the operators. Wild-type and mutant argC operators have been compared for their interaction with the repressor, using both in vivo and in vitro approaches. Our results indicate that the operators of B. stearothermophilus and T. neapolitana consist of two ARG box-like sequences, 18 bp imperfect palindromes, separated by two and three base-pairs, respectively, and that the repressors from thermophilic origin establish base-specific contacts with two major groove segments and the intervening minor groove of each ARG box, all aligned on one face of the helix. In contrast, no specific contacts are established in the minor groove facing the repressor in the centre of the operator, nevertheless this region plays a crucial structural role in complex formation, as indicated by mutant studies. This picture is reminiscent of arginine repressor binding in Escherichia coli, and therefore reinforces the uniform view of arginine regulation, but also reveals a number of striking differences at particular positions of the boxes and in the length and base-pair composition of the spacer connecting two ARG boxes in the operator. These might be responsible, in part, for subtle but important functional and mechanistic differences in the way species-specific repressors interact with their cognate target sites. These variations are underlined by the different behaviour of the repressors from E. coli, B. stearothermophilus and T. neapolitana in their potential to bind heterologous operators, their requirement for arginine, and the resistance of complex formation to non-specific competitor DNA. Our findings are discussed in view of the crystal structure of the arginine repressor from B. stearothermophilus.

Song H ，Wang H ，Gigot D ，Dimova D ，Sakanyan V ，Glansdorff N ，Charlier D ... -  《JOURNAL OF MOLECULAR BIOLOGY》

Escherichia coli purine repressor: key residues for the allosteric transition between active and inactive conformations and for interdomain signaling.

Lu F ，Brennan RG ，Zalkin H 《BIOCHEMISTRY》

Crystallographic analysis of Lac repressor bound to natural operator O1.

Previous structures of Lac repressor bound to DNA used a fully symmetric "ideal" operator sequence that is missing the central G-C base-pair present in the three natural operator sequences. Here we have determined the X-ray crystal structure of a dimeric Lac repressor bound to a 22 base-pair DNA with the natural operator O1 sequence and the anti-inducer ONPF, at 4.0 A resolution. The natural operator is bent in the same way as the symmetric sequence, due to the binding of the hinge helices of the repressor to the minor groove at the central GCGG sequence of O1. Comparison of the structures of the repressor bound to the natural and symmetric operators shows very similar overall structures, with only slight rearrangements of the headpiece domains of the repressor. Analysis of crystals with iodinated DNA shows that the operator is uniquely positioned and allows for the sequence registration of the DNA relative to the repressor to be determined. The kink in the operator is centered between the left half-site and the central G-C base-pair of O1. Our results are most consistent with a previously proposed model in which, relative to the complex with the symmetric operator, the repressor accommodates binding to the natural operator sequence by shifting the position of the right headpiece by one base-pair step towards the center of O1.

Bell CE ，Lewis M 《JOURNAL OF MOLECULAR BIOLOGY》