Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7

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 as well as its N-terminal DNA binding domain withstand crystallization. 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 a= 317 Å, diffract to 4.0 Å resolution and likely contain four PaaR2 monomers and four nanobody monomers in their asymmetric unit. Crystals of two truncates containing the N-terminal helix-turn-helix domain also interact with Nb33 and the corresponding co-crystals diffract to 1.6 and 1.75 Å resolution.

Publication year:2021

Keywords:Nanobody aided crystallography, Toxin-Antitoxin module, Transcription regulation, Transcription Factor, Structural Biology, Crystallography, Molecular Biophysics

BOF-keylabel:yes

IOF-keylabel:yes

BOF-publication weight:0.1

Authors:International

Authors from:Higher Education

Accessibility:Open