Lactose repressor hinge domain independently binds DNA

Abstract

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.

Keywords: DNA binding protein; DNA operator; DNA-protein interaction; allosteric regulation; hinge helix; lactose repressor protein; structure-function.

Figure 1

X‐ray crystallographic and NMR structures of LacI and its domains. (A) X‐ray structure for tetrameric LacI protein (PDB file 1LBG ⁴). (B) and (C) show NMR structures of DNA complexes of truncated LacI containing the N‐terminal HtH DNA binding domain and the hinge helix sequence with Val52Cys substitution. To enhance clarity, 10 of the 20 structures in the NMR‐based PDB files were deleted in these NMR structures. (B) O1 operator DNA (PDB file 1L1M 14). (C) Nonspecific DNA (PDB file 1OSL 16). The black lines indicate the position of the disulfide linkage between the hinge regions in the oxidized Val52Cys variant. Note that the complex with nonspecific DNA is linear rather than bent with no hinge helix present.

Figure 2

Purification and binding assays for His‐tagged LacI variants. (A) Purification of His₆‐LacI‐51 and His₆‐LacI‐59. Products of purification were examined using SDS‐PAGE to confirm purity (dashed boxes indicate samples used for binding assays). Lane 2 shows trypsin digestion products of wild‐type LacI (no His‐tag) for reference. (B) His‐tagged deletion mutants were used in a pull‐down assay with biotinylated O1 and O_scram DNA bound to streptavidin‐coated beads with and without IPTG (I). Note that removal of the hinge helix in His₆‐LacI‐59 results in complete loss of DNA binding. (C) Electrophoretic mobility shift assays. Varying concentrations of His₆‐LacI‐51 and 10⁻⁷ M LacI were each mixed with [32P]‐labeled 40 bp O1 DNA (≤10⁻¹¹ M) and equilibrated, followed by rapid loading onto a polyacrylamide gel. Note the loss of free DNA and detection of bound bands for His₆‐LacI‐51 at 10⁻⁵ M protein; the lowest bound band exhibits slightly higher mobility compared to wild‐type LacI, as expected for this smaller tetramer. Note that wild‐type LacI was separated in this experiment from the His₆‐LacI‐51 and O1 DNA, and the dark line indicates removal of wells that contained other materials for analysis. (D) Band intensities for free DNA were derived from phosphorimaging of four separate experiments for His₆‐LacI‐51 and three separate experiments for wild‐type LacI; the values were normalized to the free O1 DNA band intensity for each experiment. Standard deviations for free DNA in samples with bound species are indicated.

Figure 3

Nitrocellulose filter binding assays of His₆‐LacI‐51. Data were normalized to maximum binding observed to allow compilation of multiple experiments. Black‐filled circles, His₆‐LacI‐51 and O1; gray‐filled triangles, His₆‐LacI‐51, O1, and 2 mM IPTG. Open circles, His₆‐LacI‐51 and O_scram; open triangles, His₆‐LacI‐51, O_scram and 2 mM IPTG. Results are shown from four experiments for O1 binding and three experiments for O_scram binding with each experiment comprised of internal duplicate or triplicate samples. Error range corresponding to the standard deviation is shown either above or below each point to facilitate visualization (if there is no error bar, the error is smaller than the point symbol). The lines shown are fits to the data using the Hill equation (see Supporting Information for details). The protein concentration for 50% fractional saturation (R _50%) derived from the fitted data is shown in Table I for each condition as estimated equilibrium dissociation constants.

Figure 4

Hinge peptide sequence and binding data. (A) Sequence of the synthetic hinge peptide with Val52Cys substitution (arrow) to allow disulfide linkage. Amino acids in wild‐type LacI are numbered 50–62. Note that fluorescein (FL) is attached to the side chain amino group of the C‐terminal lysine residue. (B) Magnetic bead pull‐down results for synthetic hinge peptide (10 µM) in oxidized and reduced form using fluorescence to detect bound fluorescein. Data from three separate experiments with four replicates in each experiment were normalized to the average value for O1 DNA with oxidized peptide. (C), Nitrocellulose filter binding data with synthetic peptide. Values are normalized to the average value at 40 µM oxidized peptide for O1 DNA. Black bars, oxidized peptide and O1; dark gray bars, reduced peptide and O1; medium gray bars, oxidized peptide and O_scram; light gray bars, reduced peptide and O_scram. The dotted line indicating 50% of the maximum binding observed for O1 operator can be used to determine approximate binding parameters and the relative stability of the complexes.