The structural basis of differential DNA sequence recognition by restriction–modification controller proteins

Ball, Neil J.; McGeehan, J.E.; Streeter, Simon; Thresh, S.J.; Kneale, Geoff

doi:10.1093/nar/gks718

Cited by 20 publications

(32 citation statements)

References 26 publications

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“…The Y37F crystal structure has six monomers (three biological dimers) in the asymmetric unit. Five of the monomers in the asymmetric unit are observed to be in loop conformation I but the sixth more closely resembles that of conformation II, which is almost identical to that in the DNA-protein complex with the 19O M operator site [18]. Both of the R46A crystal structures and the S52A crystal structure have adopted loop conformation III, despite the varying buffer conditions and crystallographic interactions in those three structures.…”

Section: Resultsmentioning

confidence: 78%

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Structural and Mutagenic Analysis of the RM Controller Protein C.Esp1396I

2014

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Bacterial restriction-modification (RM) systems are comprised of two complementary enzymatic activities that prevent the establishment of foreign DNA in a bacterial cell: DNA methylation and DNA restriction. These two activities are tightly regulated to prevent over-methylation or auto-restriction. Many Type II RM systems employ a controller (C) protein as a transcriptional regulator for the endonuclease gene (and in some cases, the methyltransferase gene also). All high-resolution structures of C-protein/DNA-protein complexes solved to date relate to C.Esp1396I, from which the interactions of specific amino acid residues with DNA bases and/or the phosphate backbone could be observed. Here we present both structural and DNA binding data for a series of mutations to the key DNA binding residues of C.Esp1396I. Our results indicate that mutations to the backbone binding residues (Y37, S52) had a lesser affect on DNA binding affinity than mutations to those residues that bind directly to the bases (T36, R46), and the contributions of each side chain to the binding energies are compared. High-resolution X-ray crystal structures of the mutant and native proteins showed that the fold of the proteins was unaffected by the mutations, but also revealed variation in the flexible loop conformations associated with DNA sequence recognition. Since the tyrosine residue Y37 contributes to DNA bending in the native complex, we have solved the structure of the Y37F mutant protein/DNA complex by X-ray crystallography to allow us to directly compare the structure of the DNA in the mutant and native complexes.

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Section: Resultsmentioning

confidence: 78%

“…We also investigated the affinities of the protein for its three natural promoters, O M , O R and O L , in order to understand the structural and mechanistic basis of differential DNA sequence recognition in this system [18].…”

Section: Introductionmentioning

confidence: 99%

Structural and Mutagenic Analysis of the RM Controller Protein C.Esp1396I

2014

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“…S4). The A-tract region in the DNA core induces the bending of DNA through the interaction between the proteins and the phosphodiester backbone in DNA, causing compression of the minor groove (Ball et al, 2012;Hizver et al, 2001). The minorgroove width at bases 16 to 18 ($2.5 Å ) is also compressed, which stablizes the structure of MtHigA3 bound to DNA.…”

Section: Overall Structure Of Mthiga3 Bound To Dnamentioning

confidence: 99%

Induced DNA bending by unique dimerization of HigA antitoxin

Park

Kim

Pathak

et al. 2020

IUCrJ

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The bacterial toxin–antitoxin (TA) system regulates cell growth under various environmental stresses. Mycobacterium tuberculosis, the causative pathogen of tuberculosis (TB), has three HigBA type II TA systems with reverse gene organization, consisting of the toxin protein HigB and labile antitoxin protein HigA. Most type II TA modules are transcriptionally autoregulated by the antitoxin itself. In this report, we first present the crystal structure of the M. tuberculosis HigA3 antitoxin (MtHigA3) and MtHigA3 bound to its operator DNA complex. We also investigated the interaction between MtHigA3 and DNA using NMR spectroscopy. The MtHigA3 antitoxin structure is a homodimer that contains a structurally well conserved DNA-binding domain at the N-terminus and a dimerization domain at the C-terminus. Upon comparing the HigA homologue structures, a distinct difference was found in the C-terminal region that possesses the β-lid, and diverse orientations of two helix–turn–helix (HTH) motifs from HigA homologue dimers were observed. The structure of MtHigA3 bound to DNA reveals that the promoter DNA is bound to two HTH motifs of the MtHigA3 dimer presenting 46.5° bending, and the distance between the two HTH motifs of each MtHigA3 monomer was increased in MtHigA3 bound to DNA. The β-lid, which is found only in the tertiary structure of MtHigA3 among the HigA homologues, causes the formation of a tight dimerization network and leads to a unique arrangement for dimer formation that is related to the curvature of the bound DNA. This work could contribute to the understanding of the HigBA system of M. tuberculosis at the atomic level and may contribute to the development of new antibiotics for TB treatment.

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“…The affinity for the weakest operator site (O R ) is increased 100-fold when the directly adjacent O L site is occupied by a C-protein. X-ray crystallography revealed the structure of the adjacent O L and O R sites occupied by a pair of C.Esp1396I dimers forming the repression complex [1] as well as the C-protein-O M repression complex [2] and the transcriptional activation C-protein-O L complex [3]. These complexes revealed a combination of both direct and indirect readout between the C-protein and the DNA, as well as significant DNA distortion and protein-protein cooperativity.…”

Section: Bacterialmentioning

confidence: 99%