Altering Residues N125 and D149 Impacts Sugar Effector Binding and Allosteric Parameters in <i>Escherichia coli</i> Lactose Repressor

Xu, Jia; Liu, Shirley; Chen, Mingzhi; Ma, Jianpeng; Matthews, Kathleen S.

doi:10.1021/bi200896t

Cited by 13 publications

(17 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Large-scale studies on mutant libraries have helped to understand the structural features that allow LacI to be efficient at repressing transcription and specific for both inducer and operator DNA [35-37], but have rarely led to the isolation of better mutants [40]. In LacI, long range communications are responsible for the transmission of the signal from the inducer binding side to the DNA binding domain [30], as suggested by the effects of mutations in the hinge region [38] and by the introduction of disulphide bonds at positions 125, 149 and 193 [32,33]. Our results show that saturation mutagenesis at position 220 generates the non-inducible I s phenotype in half of the cases, whereas the ability to respond to IPTG is exclusive to aromatic side chains (tryptophan and phenylalanine, see Figure 2).…”

Section: Discussionmentioning

confidence: 99%

“…Binding of the inducer to the core domain stabilises the non-binding conformation, characterised by local unfolding in the DNA binding domain (up to 40 Å away) that decreases specificity for the lac operator and relieves the repression of transcription [30]. Crystallisation in the presence of both inducers and anti-inducers (IPTG and ONPF) has revealed the interaction networks in the binding pocket that correlate with either repression or de-repression [31], and mutational analysis has confirmed the importance of these amino acids [32,33]. However, the effects of random mutations [34-36] and insertions [37] in the LacI structure as well as targeted mutations in the hinge region [38] showed that the roles played by individual amino acids in long-distance allosteric transmission are still incompletely understood.…”

Section: Introductionmentioning

confidence: 99%

“…Protein engineering has brought about LacI variants with altered inducibility: directed evolution yielded variants with 100-fold tighter affinity for IPTG that carried mutations in both the DNA and inducer binding domains [40]. Similarly, mutations at positions 125 and 149 showed that changes at the inducer binding site influence the allosteric behaviour of LacI [32]. Mutants that better repress transcription were obtained by directed evolution, but required the co-evolution of a new operator DNA sequence [41].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A single mutation in the core domain of the lac repressor reduces leakiness

et al. 2013

View full text Add to dashboard Cite

BackgroundThe lac operon provides cells with the ability to switch from glucose to lactose metabolism precisely when necessary. This metabolic switch is mediated by the lac repressor (LacI), which in the absence of lactose binds to the operator DNA sequence to inhibit transcription. Allosteric rearrangements triggered by binding of the lactose isomer allolactose to the core domain of the repressor impede DNA binding and lift repression. In Nature, the ability to detect and respond to environmental conditions comes at the cost of the encoded enzymes being constitutively expressed at low levels. The readily-switched regulation provided by LacI has resulted in its widespread use for protein overexpression, and its applications in molecular biology represent early examples of synthetic biology. However, the leakiness of LacI that is essential for the natural function of the lac operon leads to an increased energetic burden, and potentially toxicity, in heterologous protein production.ResultsAnalysis of the features that confer promiscuity to the inducer-binding site of LacI identified tryptophan 220 as a target for saturation mutagenesis. We found that phenylalanine (similarly to tryptophan) affords a functional repressor that is still responsive to IPTG. Characterisation of the W220F mutant, LacIWF, by measuring the time dependence of GFP production at different IPTG concentrations and at various incubation temperatures showed a 10-fold reduction in leakiness and no decrease in GFP production. Cells harbouring a cytotoxic protein under regulatory control of LacIWF showed no decrease in viability in the early phases of cell growth. Changes in responsiveness to IPTG observed in vivo are supported by the thermal shift assay behaviour of purified LacIWF with IPTG and operator DNA.ConclusionsIn LacI, long-range communications are responsible for the transmission of the signal from the inducer binding site to the DNA binding domain and our results are consistent with the involvement of position 220 in modulating these. The mutation of this single tryptophan residue to phenylalanine generated an enhanced repressor with a 10-fold decrease in leakiness. By minimising the energetic burden and cytotoxicity caused by leakiness, LacIWF constitutes a useful switch for protein overproduction and synthetic biology.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A single mutation in the core domain of the lac repressor reduces leakiness

et al. 2013

View full text Add to dashboard Cite

show abstract

“…Nitrocellulose filter binding was used to determine binding affinities for radiolabeled O1 and O scram to His 6 -LacI, His 6 -LacI-51, and His 6 -LacI-59 and monomeric and disulfide-linked peptides. 45 The assay was conducted at room temperature in buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM KCl, 0.3 mM DTT, 0.1 mM EDTA. Samples with inducer contained 2 mM IPTG.…”

Section: Nitrocellulose Filter Binding Assaymentioning

confidence: 99%

Lactose repressor hinge domain independently binds DNA

Hewitt

Gulati

et al. 2018

Protein Science

Self Cite

View full text Add to dashboard Cite

The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ∼40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ∼4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.

show abstract

“…For E. coli LacI, structures of free, DNA-bound, and IPTG-bound protein (14,70), along with molecular dynamics simulations (23,71,72), have been used to study these adaptable regions. The largest changes are found in the linker region and in the N-subdomain interface of LacI (14,23,70).…”

Section: Allosteric Communication In the Laci/galr Proteinsmentioning

confidence: 99%