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

doi:10.1107/S2053230X21009006

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

Research output: Contribution to journal › Article › peer-review

1 Citation (Scopus)

329 Downloads (Pure)

Abstract

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 language	English
Article number	11
Pages (from-to)	374-384
Number of pages	11
Journal	Acta Crystallographica Section F - Structural Biology Communications
Volume	77
Issue number	10
DOIs	https://doi.org/10.1107/S2053230X21009006
Publication status	Published - 1 Oct 2021

Keywords

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

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10.1107/S2053230X21009006

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De Bruyn, P., Prolic Kalinsek, M., Vandervelde, A., Malfait, M., Sterckx, Y., Sobott, F., Hadzi, S., Pardon, E., Steyaert, J., & Loris, R. (2021). Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7. Acta Crystallographica Section F - Structural Biology Communications, 77(10), 374-384. Article 11. https://doi.org/10.1107/S2053230X21009006

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title = "Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7",

abstract = "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 {\AA}, diffract to 4.0 {\AA} 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 {\AA} resolution.",

keywords = "Nanobody aided crystallography, Toxin-Antitoxin module, Transcription regulation, Transcription Factor, Structural Biology, Crystallography, Molecular Biophysics",

author = "\{De Bruyn\}, Pieter and \{Prolic Kalinsek\}, Marusa and Alexandra Vandervelde and Milan Malfait and Yann Sterckx and Frank Sobott and San Hadzi and Els Pardon and Jan Steyaert and Remy Loris",

year = "2021",

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doi = "10.1107/S2053230X21009006",

language = "English",

volume = "77",

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journal = "Acta Crystallographica Section F - Structural Biology Communications",

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De Bruyn, P, Prolic Kalinsek, M, Vandervelde, A, Malfait, M, Sterckx, Y, Sobott, F, Hadzi, S, Pardon, E , Steyaert, J & Loris, R 2021, 'Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7', Acta Crystallographica Section F - Structural Biology Communications, vol. 77, no. 10, 11, pp. 374-384. https://doi.org/10.1107/S2053230X21009006

TY - JOUR

T1 - Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7

AU - De Bruyn, Pieter

AU - Prolic Kalinsek, Marusa

AU - Vandervelde, Alexandra

AU - Malfait, Milan

AU - Sterckx, Yann

AU - Sobott, Frank

AU - Hadzi, San

AU - Pardon, Els

AU - Steyaert, Jan

AU - Loris, Remy

PY - 2021/10/1

Y1 - 2021/10/1

N2 - 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.

AB - 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.

KW - Nanobody aided crystallography

KW - Toxin-Antitoxin module

KW - Transcription regulation

KW - Transcription Factor

KW - Structural Biology

KW - Crystallography

KW - Molecular Biophysics

UR - https://www.scopus.com/pages/publications/85116611532

U2 - 10.1107/S2053230X21009006

DO - 10.1107/S2053230X21009006

M3 - Article

C2 - 34605442

SN - 2053-230X

VL - 77

SP - 374

EP - 384

JO - Acta Crystallographica Section F - Structural Biology Communications

JF - Acta Crystallographica Section F - Structural Biology Communications

IS - 10

M1 - 11

ER -

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

Abstract

Keywords

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FWOAL831: Regulation of persistence by the paaAR-paaA2-parE2 and cog4197-duf1019 operons from E. coli O157

Bistable expression of a toxin-antitoxin system located in a cryptic prophage of Escherichia coliO157:H7

Prokaryote toxin-antitoxin modules: complex regulation of an unclear function

Toxin-antitoxin modules: functional characterization of transcription regulation and potential use against pathogenic bacteria

PaaR2 regulator N-terminal domain

PhD Defense Mariusa Prolic-Kalinsek - Regulatory interplay between the toxin-antitoxin cluster paaR2-paaA2-parE2 and prophage genes ydaS-ydaT from Escherichia coliO157:H7 (Event)

PhD Thesis defense Pieter De Bruyn - Toxin-antitoxin modules: functional characterization of transcription regulation and potential use against pathogenic bacteria (Event)

Cite this

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

Abstract

Keywords

Access to Document

Handle.net

Other files and links

Fingerprint

Projects

FWOAL831: Regulation of persistence by the paaAR-paaA2-parE2 and cog4197-duf1019 operons from E. coli O157

Research output

Bistable expression of a toxin-antitoxin system located in a cryptic prophage of Escherichia coliO157:H7

Prokaryote toxin-antitoxin modules: complex regulation of an unclear function

Toxin-antitoxin modules: functional characterization of transcription regulation and potential use against pathogenic bacteria

Datasets

PaaR2 regulator N-terminal domain

Activities

PhD Defense Mariusa Prolic-Kalinsek - Regulatory interplay between the toxin-antitoxin cluster paaR2-paaA2-parE2 and prophage genes ydaS-ydaT from Escherichia coliO157:H7 (Event)

PhD Thesis defense Pieter De Bruyn - Toxin-antitoxin modules: functional characterization of transcription regulation and potential use against pathogenic bacteria (Event)

Cite this