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

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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.

Originele taal-2	English
Artikelnummer	11
Pagina's (van-tot)	374-384
Aantal pagina's	11
Tijdschrift	Acta Crystallographica Section F - Structural Biology Communications
Volume	77
Nummer van het tijdschrift	10
DOI's	https://doi.org/10.1107/S2053230X21009006
Status	Published - 1 okt 2021

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10.1107/S2053230X21009006

74396672Final published version, 1,55 MBLicentie: Unspecified
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https://eprints.whiterose.ac.uk/180323/6/S2053230X21009006.pdfLicentie: Unspecified

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1 Afgelopen

FWOAL831: Regulatie van persistentie door het paaAR-paaA2-parE2 en cog4197-duf1019 operons van E. coli O157
Loris, R.
1/01/17 → 31/12/20
Project: Fundamenteel

4 Article
1 PhD Thesis

Bistable expression of a toxin-antitoxin system located in a cryptic prophage of Escherichia coliO157:H7
Jurenas, D., Fraikin, N., Goormaghtigh, F., De Bruyn, P., Vandervelde, A., Jove, T., Charlier, D., Loris, R. & Van Melderen, L., 21 dec 2021, In: mBio. 12, 6, 15 blz., e0294721.
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6 Citaten (Scopus)

32 Downloads (Pure)
Prokaryote toxin-antitoxin modules: complex regulation of an unclear function
De Bruyn, P., Girardin, Y. & Loris, R., jun 2021, In: Protein Sci. 30, 6, blz. 1103-1113 11 blz.
Onderzoeksoutput: Article › peer review

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11 Citaten (Scopus)

79 Downloads (Pure)
Toxin-antitoxin modules: functional characterization of transcription regulation and potential use against pathogenic bacteria
De Bruyn, P., 2021, Brussels.
Onderzoeksoutput: PhD Thesis

1 Member of PhD committee

Toegepaste Biologische Wetenschappen (Organisatie)
Eveline Peeters (Chair), Inge Van Molle (Jury), Kim Roelants (Jury), Remy Loris (Supervisor), Henri De Greve (Supervisor), Tanel Tenson (Jury) & Jan Michiels (Jury)
2 sep 2021
Activiteit: Member of PhD committee

Citeer dit

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. [11]. https://doi.org/10.1107/S2053230X21009006

De Bruyn, Pieter ; Prolic Kalinsek, Marusa ; Vandervelde, Alexandra ; Malfait, Milan ; Sterckx, Yann ; Sobott, Frank ; Hadzi, San ; Pardon, Els ; Steyaert, Jan ; Loris, Remy. / Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7. In: Acta Crystallographica Section F - Structural Biology Communications. 2021 ; Vol. 77, Nr. 10. blz. 374-384.

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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.",

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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",

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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, nr. 10, 11, blz. 374-384. https://doi.org/10.1107/S2053230X21009006

Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7. / De Bruyn, Pieter ; Prolic Kalinsek, Marusa; Vandervelde, Alexandra; Malfait, Milan; Sterckx, Yann; Sobott, Frank; Hadzi, San; Pardon, Els ; Steyaert, Jan ; Loris, Remy.

In: Acta Crystallographica Section F - Structural Biology Communications, Vol. 77, Nr. 10, 11, 01.10.2021, blz. 374-384.

Onderzoeksoutput: Article › peer review

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.

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KW - Transcription Factor

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KW - Crystallography

KW - Molecular Biophysics

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U2 - 10.1107/S2053230X21009006

DO - 10.1107/S2053230X21009006

M3 - Article

C2 - 34605442

VL - 77

SP - 374

EP - 384

JO - Acta Crystallographica Section F - Structural Biology Communications

JF - Acta Crystallographica Section F - Structural Biology Communications

SN - 2053-230X

IS - 10

M1 - 11

ER -

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

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Projecten

FWOAL831: Regulatie van persistentie door het paaAR-paaA2-parE2 en cog4197-duf1019 operons van E. coli O157

Onderzoekersoutput

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

Activiteiten

Toegepaste Biologische Wetenschappen (Organisatie)

Citeer dit