Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus

Anita Y. Lam; Els Pardon; Konstantin V. Korotkov; Wim G.J. Hol; Jan Steyaert

doi:10.1016/j.jsb.2008.11.008

Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus

Anita Y. Lam
, Els Pardon
, Konstantin V. Korotkov
, Wim G.J. Hol
, Jan Steyaert

Producción científica: Article › revisión exhaustiva

69 Citas (Scopus)

Resumen

Pseudopilins form the central pseudopilus of the sophisticated bacterial type 2 secretion systems. The crystallization of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus was greatly accelerated by the use of nanobodies, which are the smallest antigen-binding fragments derived from heavy-chain only camelid antibodies. Seven anti-EpsI:EpsJ nanobodies were generated and co-crystallization of EpsI:EpsJ nanobody complexes yielded several crystal forms very rapidly. In the structure solved, the nanobodies are arranged in planes throughout the crystal lattice, linking layers of EpsI:EpsJ heterodimers. The EpsI:EpsJ dimer observed confirms a right-handed architecture of the pseudopilus, but, compared to a previous structure of the EpsI:EpsJ heterodimer, EpsI differs 6° in orientation with respect to EpsJ; one loop of EpsJ is shifted by ∼5 Å due to interactions with the nanobody; and a second loop of EpsJ underwent a major change of 17 Å without contacts with the nanobody. Clearly, nanobodies accelerate dramatically the crystallization of recalcitrant protein complexes and can reveal conformational flexibility not observed before.

Idioma original	English
Páginas (desde-hasta)	8-15
Número de páginas	8
Publicación	Journal of Structural Biology
Volumen	166
N.º	1
DOI	https://doi.org/10.1016/j.jsb.2008.11.008
Estado	Published - abr 2009

Nota bibliográfica

Funding Information:
We thank Jürgen Bosch, Stewart Turley, and Jan Abendroth for help and valuable discussions. We thank Nele Buys for the selection, expression and purification of the nanobodies. We are indebted to the support staff of beamline 9-2 of the SSRL for assistance during data collection. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), supported by the Department of Energy and by NIH. This work was supported by Grant AI34501 from the NIH, by the Howard Hughes Medical Institute (HHMI) to W.G.J.H., and University of Washington’s Molecular Biophysics Training Grant (5 T32 GM008268-19) from the NIH. This work was also supported by the Belgian Government under the framework of the Interuniversity Attraction Poles (I.A.P. P6/19).

Financiación

We thank Jürgen Bosch, Stewart Turley, and Jan Abendroth for help and valuable discussions. We thank Nele Buys for the selection, expression and purification of the nanobodies. We are indebted to the support staff of beamline 9-2 of the SSRL for assistance during data collection. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), supported by the Department of Energy and by NIH. This work was supported by Grant AI34501 from the NIH, by the Howard Hughes Medical Institute (HHMI) to W.G.J.H., and University of Washington’s Molecular Biophysics Training Grant (5 T32 GM008268-19) from the NIH. This work was also supported by the Belgian Government under the framework of the Interuniversity Attraction Poles (I.A.P. P6/19).

Financiadores	Número del financiador
National Institutes of Health (NIH)	AI34501
Howard Hughes Medical Institute
U.S. Department of Energy EPSCoR
National Institute of General Medical Sciences DP2GM119177 Sophie Dumont National Institute of General Medical Sciences	T32GM008268
The George Washington University	P6/19, 5 T32 GM008268-19

ASJC Scopus subject areas

Structural Biology

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10.1016/j.jsb.2008.11.008

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title = "Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus",

abstract = "Pseudopilins form the central pseudopilus of the sophisticated bacterial type 2 secretion systems. The crystallization of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus was greatly accelerated by the use of nanobodies, which are the smallest antigen-binding fragments derived from heavy-chain only camelid antibodies. Seven anti-EpsI:EpsJ nanobodies were generated and co-crystallization of EpsI:EpsJ nanobody complexes yielded several crystal forms very rapidly. In the structure solved, the nanobodies are arranged in planes throughout the crystal lattice, linking layers of EpsI:EpsJ heterodimers. The EpsI:EpsJ dimer observed confirms a right-handed architecture of the pseudopilus, but, compared to a previous structure of the EpsI:EpsJ heterodimer, EpsI differs 6° in orientation with respect to EpsJ; one loop of EpsJ is shifted by ∼5 {\AA} due to interactions with the nanobody; and a second loop of EpsJ underwent a major change of 17 {\AA} without contacts with the nanobody. Clearly, nanobodies accelerate dramatically the crystallization of recalcitrant protein complexes and can reveal conformational flexibility not observed before.",

keywords = "Crystallization chaperones, General secretory pathway, Pseudopilins, Single-chain antibody",

author = "Lam, \{Anita Y.\} and Els Pardon and Korotkov, \{Konstantin V.\} and Hol, \{Wim G.J.\} and Jan Steyaert",

note = "Funding Information: We thank J{\"u}rgen Bosch, Stewart Turley, and Jan Abendroth for help and valuable discussions. We thank Nele Buys for the selection, expression and purification of the nanobodies. We are indebted to the support staff of beamline 9-2 of the SSRL for assistance during data collection. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), supported by the Department of Energy and by NIH. This work was supported by Grant AI34501 from the NIH, by the Howard Hughes Medical Institute (HHMI) to W.G.J.H., and University of Washington{\textquoteright}s Molecular Biophysics Training Grant (5 T32 GM008268-19) from the NIH. This work was also supported by the Belgian Government under the framework of the Interuniversity Attraction Poles (I.A.P. P6/19). ",

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doi = "10.1016/j.jsb.2008.11.008",

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AU - Lam, Anita Y.

AU - Pardon, Els

AU - Korotkov, Konstantin V.

AU - Hol, Wim G.J.

AU - Steyaert, Jan

N1 - Funding Information: We thank Jürgen Bosch, Stewart Turley, and Jan Abendroth for help and valuable discussions. We thank Nele Buys for the selection, expression and purification of the nanobodies. We are indebted to the support staff of beamline 9-2 of the SSRL for assistance during data collection. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), supported by the Department of Energy and by NIH. This work was supported by Grant AI34501 from the NIH, by the Howard Hughes Medical Institute (HHMI) to W.G.J.H., and University of Washington’s Molecular Biophysics Training Grant (5 T32 GM008268-19) from the NIH. This work was also supported by the Belgian Government under the framework of the Interuniversity Attraction Poles (I.A.P. P6/19).

PY - 2009/4

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N2 - Pseudopilins form the central pseudopilus of the sophisticated bacterial type 2 secretion systems. The crystallization of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus was greatly accelerated by the use of nanobodies, which are the smallest antigen-binding fragments derived from heavy-chain only camelid antibodies. Seven anti-EpsI:EpsJ nanobodies were generated and co-crystallization of EpsI:EpsJ nanobody complexes yielded several crystal forms very rapidly. In the structure solved, the nanobodies are arranged in planes throughout the crystal lattice, linking layers of EpsI:EpsJ heterodimers. The EpsI:EpsJ dimer observed confirms a right-handed architecture of the pseudopilus, but, compared to a previous structure of the EpsI:EpsJ heterodimer, EpsI differs 6° in orientation with respect to EpsJ; one loop of EpsJ is shifted by ∼5 Å due to interactions with the nanobody; and a second loop of EpsJ underwent a major change of 17 Å without contacts with the nanobody. Clearly, nanobodies accelerate dramatically the crystallization of recalcitrant protein complexes and can reveal conformational flexibility not observed before.

AB - Pseudopilins form the central pseudopilus of the sophisticated bacterial type 2 secretion systems. The crystallization of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus was greatly accelerated by the use of nanobodies, which are the smallest antigen-binding fragments derived from heavy-chain only camelid antibodies. Seven anti-EpsI:EpsJ nanobodies were generated and co-crystallization of EpsI:EpsJ nanobody complexes yielded several crystal forms very rapidly. In the structure solved, the nanobodies are arranged in planes throughout the crystal lattice, linking layers of EpsI:EpsJ heterodimers. The EpsI:EpsJ dimer observed confirms a right-handed architecture of the pseudopilus, but, compared to a previous structure of the EpsI:EpsJ heterodimer, EpsI differs 6° in orientation with respect to EpsJ; one loop of EpsJ is shifted by ∼5 Å due to interactions with the nanobody; and a second loop of EpsJ underwent a major change of 17 Å without contacts with the nanobody. Clearly, nanobodies accelerate dramatically the crystallization of recalcitrant protein complexes and can reveal conformational flexibility not observed before.

KW - Crystallization chaperones

KW - General secretory pathway

KW - Pseudopilins

KW - Single-chain antibody

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JO - Journal of Structural Biology

JF - Journal of Structural Biology

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Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus

Resumen

Nota bibliográfica

Financiación

ASJC Scopus subject areas

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