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

Pieter De Bruyn, Marusa Prolic Kalinsek, Alexandra Vandervelde, Milan Malfait, Yann Sterckx, Frank Sobott, San Hadzi, Els Pardon, Jan Steyaert, Remy Loris

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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.
Original languageEnglish
Article number11
Pages (from-to)374-384
Number of pages11
JournalActa Crystallographica Section F - Structural Biology Communications
Issue number10
Publication statusPublished - 1 Oct 2021


  • Nanobody aided crystallography
  • Toxin-Antitoxin module
  • Transcription regulation
  • Transcription Factor
  • Structural Biology
  • Crystallography
  • Molecular Biophysics


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