A closer view of the conformation of the Lac repressor bound to operator

Crystal structures of the Lac repressor, with and without isopropyithiogalactoside (IPTG), and the repressor bound to operator have provided a model for how the binding of the inducer reduces the affinity of the repressor for the operator. However, because of the low resolution of the operator-bound structure (4.8 A), the model for the allosteric transition was presented in terms of structural elements rather than in terms of side chain interactions. Here we have constructed a dimeric Lac repressor and determined its structure at 2.6 A resolution in complex with a symmetric operator and the anti-inducer orthonitrophenylfucoside (ONPF). The structure enables the induced (IPTG-bound) and repressed (operator-bound) conformations of the repressor to be compared in atomic detail. An extensive network of interactions between the DNA-binding and core domains of the repressor suggests a possible mechanism for the allosteric transition.     

How Lac repressor finds lac operator in vitro.

Filter-binding and gel mobility shift assays were used to analyse the kinetics of the interaction of Lac repressor with lac operator. A comparison of the two techniques reveals that filter-binding assays with tetrameric Lac repressor have often been misinterpreted. It has been assumed that all complexes of Lac repressor and lac operator DNA bind with equal affinity to nitrocellulose filters. This assumption is wrong. Sandwich or loop complexes where two lac operators bind to one tetrameric Lac repressor are not or are only badly retained on nitrocellulose filters under normal conditions. Taking this into account, dimeric and tetrameric Lac repressor do not show any DNA-length dependence of their association and dissociation rate constants when they bind to DNA fragments smaller than 2455 base-pairs carrying a single symmetric ideal lac operator. A ninefold increased association rate to ideal lac operator on lambda DNA is observed for tetrameric but not dimeric Lac repressor. It is presumably due to intersegment transfer involving lac operator-like sequences.     

Arc repressor is tetrameric when bound to operator DNA

The Arc repressor of bacteriophage P22 is a member of a family of DNA-binding proteins that use N-terminal residues in a beta-sheet conformation for operator recognition. Here, Arc is shown to bind to its operator site as a tetramer. When mixtures of Arc (53 residues) and an active variant of Arc (78 residues) are used in gel retardation experiments, five discrete protein-DNA complexes are observed. This result is as expected for operators bearing heterotetramers containing 4:0, 3:1, 2:2, 1:3, and 0:4 ratios of the two proteins. Direct measurements of binding stoichiometry support the conclusion that Arc binds to a single 21-base-pair operator site as a tetramer. The Arc-operator binding reaction is highly cooperative (Hill constant = 3.5) and involves at least two coupled equilibria. In the first reaction, two unfolded monomers interact to form a folded dimer (Bowie & Sauer, 1989a). Rapid dilution experiments indicate that the Arc dimer is the kinetically significant DNA-binding species and allow an estimate of the equilibrium dissociation constant for dimerization [K1 = 5 (+/- 3) x 10(-9) M]. The rate of association of Arc-operator complexes shows the expected second-order dependence on the concentration of free Arc dimers, with k2 = 2.8 (+/- 0.7) x 10(18) M-2 s-1. The dissociation of Arc-operator complexes is a first-order process with k-2 = 1.6 (+/- 0.6) x 10(-4) s-1. The ratio of these kinetic constants [K2 = 5.7 (+/- 2.3) x 10(-23) M2] provides an estimate for the equilibrium constant for dissociation of the DNA-bound tetramer to two free Arc dimers and the operator. An independent determination of this complex equilibrium constant [K2 = 7.8 (+/- 4.8) x 10(-23) M2] was obtained from equilibrium binding experiments.     

Model of a LexA repressor dimer bound to recA operator

A complete three dimensional model (RCSB000408; PDB code 1qaa) for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor is proposed. A model of interaction of the N-terminal operator binding domain 1-72 with the operator was available. We have modelled residues 106-202 of LexA on the basis of the crystal structure of a homologous protein, UmuD'. Residues 70-105 have been modelled by us, residues 70-77 comprising the real hinge, followed by a beta-strand and an alpha-helix, both interacting with the rest of the C-domain. The preexponential Arrhenius factor for the LexA autocleavage is shown to be approximately 10(9) s(-1) at 298K whereas the exponential factor is approximately 2 x 10(-12), demanding that the autocleavage site is quite close to the catalytic site but reaction is slow due to an activation energy barrier. We propose that in the operator bound form, Ala 84- Gly 85 is about 7-10A from the catalytic groups, but the reaction does not occur as the geometry is not suitable for a nucleophilic attack from Ser 119 Ogamma, since Pro 87 is held in the cis conformation. When pH is elevated or under the action of activated RecA, cleavage may occur following a cis --> trans isomerization at Pro 87 and/or a rotation of the region beta9-beta10 about beta7-beta8 following the disruption of two hydrogen bonds. We show that the C-C interaction comprises the approach of two negatively charged surfaces neutralized by sodium ions, the C-domains of the monomers making a new beta barrel at the interface burying 710A2 of total surface area of each monomer.     

Lac repressor with the helix-turn-helix motif of lambda cro binds to lac operator.

Lac repressor, lambda cro protein and their operator complexes are structurally, biochemically and genetically well analysed. Both proteins contain a helix-turn-helix (HTH) motif which they use to bind specifically to their operators. The DNA sequences 5'-GTGA-3' and 5'-TCAC-3' recognized in palindromic lac operator are the same as in lambda operator but their order is inverted form head to head to tail to tail. Different modes of aggregation of the monomers of the two proteins determine the different arrangements of the HTH motifs. Here we show that the HTH motif of lambda cro protein can replace the HTH motif of Lac repressor without changing its specificity. Such hybrid Lac repressor is unstable. It binds in vitro more weakly than Lac repressor but with the same specificity to ideal lac operator. It does not bind to consensus lambda operator.     

Lac repressor - lac operator interaction. Circular dichroism study.

The interaction between lac repressor and a small operator DNA fragment have been examined by circular dichroism spectroscopy. The binding of lac repressor on the operator induces a conformation change of the DNA which is different from that observed upon non specific binding on non operator DNA. The CD titration curve indicates that the stoechiometry of interaction is complex. A two operators-one repressor complex was found. This result was confirmed by a gel filtration experiment.     

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.     

Improved model of a LexA repressor dimer bound to recA operator

A complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Delta(1-67) S119A, S119A, G85D and Delta(1-67) quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the 'cleavable' cleavage site region found in the crystal structures is actually the more relevant form of the region in wild-type LexA since it agrees with the value of the pre-exponential Arrhenius factor for its autocleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a 'conformational switch' inferred from the crystal structures is meaningless.     

Crystallographic analysis of triclosan bound to enoyl reductase

Molecular genetic studies with strains of Escherichia coli resistant to triclosan, an ingredient of many anti-bacterial household goods, have suggested that this compound works by acting as an inhibitor of enoyl reductase (ENR) and thereby blocking lipid biosynthesis. We present structural analyses correlated with inhibition data, on the complexes of E. coli and Brassica napus ENR with triclosan and NAD(+) which reveal how triclosan acts as a site-directed, picomolar inhibitor of the enzyme by mimicking its natural substrate. Elements of both the protein and the nucleotide cofactor play important roles in triclosan recognition, providing an explanation for the factors controlling its tight binding to the enzyme and for the emergence of triclosan resistance.     

Single and double loop formation when deoR repressor binds to its natural operator sites

Distal effects on the in vivo repression of the deo operon are thought to be mediated by the deoR repressor with DNA loop formation. Such loops are easily observed by electron microscopy when the oligomeric deoR repressor is added to a DNA fragment carrying the three genetically defined operators at their chromosomal distances. Upon binding of deoR to any two operators, single loops are formed, 280, 600, and 880 bp in size. With the deo operon, double loops are also formed, which are the combination of the 280 bp and 600 bp loops and the result of simultaneous binding of the protein to its three sites. The formation of both single and double loops is consistent with the long-range effects observed in vivo and with the cooperative involvement of all three operator sites in the repression.     

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.     

Crystallographic analysis of transition-state mimics bound to penicillopepsin: phosphorus-containing peptide analogues.

The molecular structures of three phosphorus-based peptide inhibitors of aspartyl proteinases complexed with penicillopepsin [1, Iva-L-Val-L-Val-StaPOEt [Iva = isovaleryl, StaP = the phosphinic acid analogue of statine [(S)-4-amino-(S)-3-hydroxy-6-methylheptanoic acid] (IvaVVStaPOEt)]; 2, Iva-L-Val-L-Val-L-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid analogue of L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = methyl ester] [Iva VVLP(O)FOMe]; and 3, Cbz-L-Ala-L-Ala-L-LeuP-(O)-Phe-OMe (Cbz = benzyloxycarbonyl) [CbzAALP(O)FOMe]] have been determined by X-ray crystallography and refined to crystallographic agreement factors, R ( = sigma parallel to F0 magnitude of - Fc parallel to/sigma magnitude of F0), of 0.132, 0.131, and 0.134, respectively. These inhibitors were designed to be structural mimics of the tetrahederal transition-state intermediate encountered during aspartic proteinase catalysis. They are potent inhibitors of penicillopepsin with Ki values of 1, 22 nM; 2, 2.8 nM; and 3, 1600 nM, respectively [Bartlett, P. A., Hanson, J. E., & Giannousis, P. P. (1990) J. Org. Chem. 55, 6268-6274]. All three of these phosphorus-based inhibitors bind virtually identically in the active site of penicillopepsin in a manner that closely approximates that expected for the transition state [James, M. N. G., Sielecki, A.R., Hayakawa, K., & Gelb, M. H. (1992) Biochemistry 31, 3872-3886]. The pro-S oxygen atom of the two phosphonate inhibitors and of the phosphinate group of the StaP inhibitor make very short contact distances (approximately 2.4 A) to the carboxyl oxygen atom, O delta 1, of Asp33 on penicillopepsin. We have interpreted this distance and the stereochemical environment of the carboxyl and phosphonate groups in terms of a hydrogen bond that most probably has a symmetric single-well potential energy function. The pro-R oxygen atom is the recipient of a hydrogen bond from the carboxyl group of Asp213. Thus, we are able to assign a neutral status to Asp213 and a partially negatively charged status to Asp33 with reasonable confidence. Similar very short hydrogen bonds involving the active site glutamic acid residues of thermolysin and carboxypeptidase A and the pro-R oxygen of bound phosphonate inhibitors have been reported [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., & Matthews, B. W. (1987) Biochemistry 26, 8542-8553; Kim, H., & Lipscomb, W. N. (1991) Biochemistry 30, 8171-8180].(ABSTRACT TRUNCATED AT 400 WORDS)     

"Cold-sensitive" mutants of the Lac repressor

Thirteen of more than 4,000 single-amino-acid-replacement mutants of the Lac repressor, generated by suppression of amber nonsense mutants, were characterized as having a cold-sensitive phenotype. However, when expressed as missense mutations, none of the replacements cause cold sensitivity, implicating the suppression mechanism as being responsible for this phenotype.     

Dimerisation mutants of Lac repressor. I. A monomeric mutant, L251A, that binds Lac operator DNA as a dimer.

Dimer formation between monomers of the Escherichia coli Lac repressor is substantially specificed by the interactions between three alpha-helices in each monomer which form a hydrophobic interface. As a first step in analysing the specificity of this interaction, we examined the mutant L251A. LacR bearing this mutation in a background lacking the C-terminal heptad repeats is completely incapable of forming dimers in solution, with a dimer-monomer equilibrium dissociation constant, or Kd, higher than 10(-5)M. This correlates with a 200-fold decrease in its ability to repress the lac operon in vivo compared to dimeric LacR. Surprisingly, the mutant is still capable of forming dimers upon binding to short operator DNA in vitro. Analysis of the kinetic parameters of binding of the mutant to operator DNA reveals a 2000 to 3000-fold increase in the equilibrium dissociation constant (Kd) of the mutant-DNA complex in comparison to dimeric LacR-operator complexes, with the change almost entirely due to a greater than 1000-fold decrease in association rate. The dissociation rate varies only by a factor of about two, in comparison to dimeric LacR. This change reflects a kinetic pathway in which dimer formation, in solution or on DNA, is the rate-limiting step. These findings have implications for the specificity and stability of the protein-protein interface in question.     

A comparative three-dimensional model of the carboxy-terminal domain of the lambda repressor and its use to build intact repressor tetramer models bound to adjacent operator sites

A model for residues 93-236 of the lambda repressor (1gfx) was predicted, based on the UmuD(') crystal structure, as part of four intact repressor molecules bound to two adjacent operator sites. The structure of region 136-230 in 1gfx was found to be nearly identical to the independently determined crystal structure of the 132-236 fragment, 1f39, released later by the PDB. Later, two more tetrameric models of the lambda repressor tetramer bound to two adjacent operator sites were constructed by us; in one of these, 1j5g, the N-domain and C-domain coordinates and hence monomer-monomer and dimer-dimer interactions are almost the same as in 1gfx, but the structure of the linker region is partly based on the linker region of the LexA dimer in 1jhe; in the other, 1lwq, the crystalline tetramer for region 140-236 has been coopted from the crystal structure deposited in 1kca, the operator DNA and N-domain coordinates of which are same as those in 1gfx and 1j5g, but the linker region is partly based on the LexA dimer structures 1jhe and 1jhh. Monomer-monomer interactions at the same operator site are stabilized by exposed hydrophobic side chains in beta-strands while cooperative interactions are mostly confined to beta(6) and some adjacent residues in both 1gfx and 1j5g. Mutational data, existence of a twofold axis relating two C-domains within a dimer, and minimization of DNA distortion between adjacent operator sites allow us to roughly position the C-domain with respect to the N-domain for both 1gfx and 1j5g. The study correlates these models with functional, biochemical, biophysical, and immunological data on the repressor in the literature. The oligomerization mode observed in the crystal structure of 132-236 may not exist in the intact repressor bound to the operator since it is shown to contradict several published biochemical data on the intact repressor.