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 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.
| Original language | English |
|---|---|
| Pages (from-to) | 8-15 |
| Number of pages | 8 |
| Journal | Journal of Structural Biology |
| Volume | 166 |
| Issue number | 1 |
| DOIs | |
| State | Published - Apr 2009 |
Bibliographical note
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).
Funding
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).
| Funders | Funder number |
|---|---|
| National Institutes of Health (NIH) | AI34501 |
| Howard Hughes Medical Institute | |
| Michigan State University-U.S. Department of Energy (MSU-DOE) Plant Research Laboratory | |
| National Institute of General Medical Sciences | T32GM008268 |
| The George Washington University | P6/19, 5 T32 GM008268-19 |
Keywords
- Crystallization chaperones
- General secretory pathway
- Pseudopilins
- Single-chain antibody
ASJC Scopus subject areas
- Structural Biology