ABSTRACT
YdaT is a functional equivalent of the CII repressor in certain lambdoid phages and prophages. YdaT from the cryptic prophage CP-933P in the genome of Escherichia coli O157:H7 is functional as a DNA-binding protein and recognizes a 5'-TTGATTN6AATCAA-3' inverted repeat. The DNA-binding domain is a helix-turn-helix (HTH)-containing POU domain and is followed by a long α-helix (α6) that forms an antiparallel four-helix bundle, creating a tetramer. The loop between helix α2 and the recognition helix α3 in the HTH motif is unusually long compared with typical HTH motifs, and is highly variable in sequence and length within the YdaT family. The POU domains have a large degree of freedom to move relative to the helix bundle in the free structure, but their orientation becomes fixed upon DNA binding.
Subject(s)
Escherichia coli O157 , Prophages , DNA-Binding Proteins , Protein Domains , DNAABSTRACT
paaR2-paaA2-parE2 is a three-component toxin-antitoxin module found in prophage CP-993P of Escherichia coli O157:H7. Transcription regulation of this module occurs via the 123-amino-acid regulator PaaR2, which forms a large oligomeric structure. Despite appearing to be well folded, PaaR2 withstands crystallization, as does its N-terminal DNA-binding domain. Native mass spectrometry was used to screen for nanobodies that form a unique complex and stabilize the octameric structure of PaaR2. One such nanobody, Nb33, allowed crystallization of the protein. The resulting crystals belong to space group F432, with unit-cell parameter a = 317â Å, diffract to 4.0â Å resolution and are likely to contain four PaaR2 monomers and four nanobody monomers in the asymmetric unit. Crystals of two truncates containing the N-terminal helix-turn-helix domain also interact with Nb33, and the corresponding co-crystals diffracted to 1.6 and 1.75â Å resolution.
Subject(s)
Crystallography, X-Ray/methods , Escherichia coli O157/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Single-Domain Antibodies/metabolism , Amino Acid Sequence , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Models, Molecular , Protein Conformation , Sequence Homology , Single-Domain Antibodies/chemistryABSTRACT
The cryptic prophage CP-933P in Escherichia coli O157:H7 contains a parDE-like toxin-antitoxin module, the operator region of which is recognized by two flanking transcription regulators: PaaR2 (ParE associated Regulator), which forms part of the paaR2-paaA2-parE2 toxin-antitoxin operon and YdaS (COG4197), which is encoded in the opposite direction but shares the operator. Here we report the 1H, 15N and 13C backbone and side chain chemical shift assignments of YdaS from Escherichia coli O157:H7 in its free state. YdaS is a distinct relative to HigA antitoxins but behaves as a monomer in solution. The BMRB Accession Number is 27917.
Subject(s)
Carbon-13 Magnetic Resonance Spectroscopy , DNA-Binding Proteins/chemistry , Escherichia coli O157/metabolism , Escherichia coli Proteins/chemistry , Proton Magnetic Resonance Spectroscopy , Nitrogen Isotopes/chemistry , Protein Structure, SecondaryABSTRACT
PaaR2 is a putative transcription regulator encoded by a three-component parDE-like toxin-antitoxin module from Escherichia coli O157:H7. Although this module's toxin, antitoxin, and toxin-antitoxin complex have been more thoroughly investigated, little remains known about its transcription regulator PaaR2. Using a wide range of biophysical techniques (circular dichroism spectroscopy, size-exclusion chromatography-multiangle laser light scattering, dynamic light scattering, small-angle x-ray scattering, and native mass spectrometry), we demonstrate that PaaR2 mainly consists of α-helices and displays a concentration-dependent octameric build-up in solution and that this octamer contains a global shape that is significantly nonspherical. Thermal unfolding of PaaR2 is reversible and displays several transitions, suggesting a complex unfolding mechanism. The unfolding data obtained from spectroscopic and calorimetric methods were combined into a unifying thermodynamic model, which suggests a five-state unfolding trajectory. Furthermore, the model allows the calculation of a stability phase diagram, which shows that, under physiological conditions, PaaR2 mainly exists as a dimer that can swiftly oligomerize into an octamer depending on local protein concentrations. These findings, based on a thorough biophysical and thermodynamic analysis of PaaR2, may provide important insights into biological function such as DNA binding and transcriptional regulation.
