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Dimer interface of Deinococcus radiodurans HucR communicates DNA and ligand binding
Author(s) -
DEOCHAND DINESH KUMAR,
Perera Inoka Chinthana,
Grove Anne
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.550.2
Subject(s) - cooperativity , deinococcus radiodurans , dimer , chemistry , histidine , ligand (biochemistry) , dna , mutant , dna binding domain , promoter , crystallography , cooperative binding , binding site , stereochemistry , biophysics , biochemistry , gene , amino acid , biology , transcription factor , gene expression , receptor , organic chemistry
Deinococcus radiodurans HucR (Hypothetical Uricase Regulator) is a transcriptional regulator belonging to the MarR family of proteins. HucR binds to promoters of hucr and uricase genes leading to repression of both. In presence of the ligand urate, the repression of both genes is relieved. The crystal structure of HucR reveals that it is a dimer, each monomer with the topology α1‐α2‐β1‐α4‐α5‐β2‐β3‐α6‐α7. The framework of the dimerization domain is provided by α2/α2′, in which the imidazole rings of His51 and His51′ are stacked. DNA binding affinity assessed by EMSA for HucR and its mutant H51F at pH 8.0 and 5.0 shows that the HucR‐H51F mutant is less sensitive to pH. Analysis of tryptophan intrinsic fluorescence spectra shows that HucR binds urate with negative cooperativity whereas HucR‐H51F binds with positive cooperativity. Moreover, thermal shift assays of HucR and HucR‐H51F demonstrate that HucR is completely unfolded at pH 5.0 and has a T m ~ 52 °C at pH 8.0 while HucR‐H51F is only modestly destabilized at pH 5.0 (T m ~ 45 °C). Notably, DNA binding stabilizes HucR at pH 5.0 whereas HucR‐H51 is destabilized on binding DNA. We suggest that histidine residues at the dimer interface act as a pH sensor and that DNA and ligand binding is communicated through the dimer interface.