Lactose Repressor Hinge Domain Independently Binds Dna
PROTEIN SCIENCE(2018)
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 approximate to 40 degrees 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 approximate to 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.
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Key words
allosteric regulation,DNA binding protein,DNA operator,DNA-protein interaction,hinge helix,lactose repressor protein,structure-function
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