|Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus|Lam, A.Y.; Pardon, E.; Korotkov, K.V.; Hol, W.G.J.; Steyaert, J. (2009). Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus. J. Struct. Biol. 166(1): 8-15. dx.doi.org/10.1016/j.jsb.2008.11.008
In: Journal of structural biology. ACADEMIC PRESS INC ELSEVIER SCIENCE: San Diego, Calif.. ISSN 1047-8477, more
General secretory pathway; Pseudopilins; Single-chain antibody;Crystallization chaperones
|Authors|| || Top |
- Lam, A.Y.
- Pardon, E.
- Korotkov, K.V.
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.