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.

Long Time-Scale Atomistic Simulations of the Structure and Dynamics of Transcription Factor-DNA Recognition

“…This analysis shows that the binding domain is indeed flexible, with a variety of conformations sampled during the simulation. In addition, in agreement with previous studies, 87 it appears that the DNA binding helices are capable of moving independently of each other, which likely affects the search process on the DNA. This is particularly interesting, because the hinge sequence itself may also contribute substantially to the binding affinity for both specific and nonspecific DNA, while also playing an important role in the transition from nonspecific to specific binding.…”

“…The observed changes in the hinge helices in the simulations of free LacI are of interest, as the HTH motif observed in LacI (Figure ) is a common feature of many DNA-binding proteins, , and the formation of the hinge helices has been argued to be an important feature for DNA bending in various lac repressor-operator complexes. , It has also been argued that the protein–DNA interface of the nonspecific complex is flexible and that this assists in rapid and efficient search for the target site on the DNA . In addition, a recent study has shown that the LacI hinge domain independently binds DNA, with the hinge region mediating both specific and nonspecific binding, with only small differences in affinity between the two. Following from this, computational studies have suggested that the hinge region plays an important contribution to the electrostatic energy of the LacI-DNA complex, the salt dependence of this electrostatic energy, and the number of salt ions excluded as the LacI-DNA complex is formed and that the binding of the hinge region of the lac repressor to DNA perturbs networks of correlated motions in the protein that in turn regulates its DNA binding affinity …”

“…83,86 It has also been argued that the protein−DNA interface of the nonspecific complex is flexible and that this assists in rapid and efficient search for the target site on the DNA. 12 In addition, a recent study has shown that the LacI hinge domain independently binds DNA, 87 with the hinge region mediating both specific and nonspecific binding, 87 with only small differences in affinity between the two. Following from this, computational studies have suggested that the hinge region plays an important contribution to the electrostatic energy of the LacI-DNA complex, the salt dependence of this electrostatic energy, and the number of salt ions excluded as the LacI-DNA complex is 52 For the corresponding pernucleotide values, see Figure S3, and for representative DNA conformations see Figure S4.…”

“…This is particularly interesting, because the hinge sequence itself may also contribute substantially to the binding affinity for both specific and nonspecific DNA, while also playing an important role in the transition from nonspecific to specific binding. 86,87 Simulations of Free DNA in the Absence of LacI. A second natural point to consider is the extent to which the intrinsic behavior of the DNA itself determines differences in LacI-DNA recognition.…”

Long Time-Scale Atomistic Simulations of the Structure and Dynamics of Transcription Factor-DNA Recognition

“…83,86 It has also been argued that the protein−DNA interface of the nonspecific complex is flexible and that this assists in rapid and efficient search for the target site on the DNA. 12 In addition, a recent study has shown that the LacI hinge domain independently binds DNA, 87 with the hinge region mediating both specific and nonspecific binding, 87 with only small differences in affinity between the two. Following from this, computational studies have suggested that the hinge region plays an important contribution to the electrostatic energy of the LacI-DNA complex, the salt dependence of this electrostatic energy, and the number of salt ions excluded as the LacI-DNA complex is These values are average values over all 80 nucleotides based on the standard A-DNA and B-DNA forms of the O SymL operator sequence, as generated by the 3DNA web server.…”