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

TitleNanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus.
Publication TypeJournal Article
Year of Publication2009
AuthorsLam, A. Y., E. Pardon, K. V. Korotkov, W. G. J. Hol, and J. Steyaert
JournalJ Struct Biol
Volume166
Issue1
Pagination8-15
Date Published2009 Apr
ISSN1095-8657
KeywordsAmino Acid Sequence, Animals, Antibodies, Monoclonal, Antigen-Antibody Complex, Binding Sites, Antibody, Camelids, New World, Complementarity Determining Regions, Crystallization, Crystallography, X-Ray, Epitopes, Immunoglobulin Heavy Chains, Membrane Transport Proteins, Models, Molecular, Molecular Sequence Data, Protein Multimerization, Protein Structure, Quaternary, Recombinant Proteins, Sequence Alignment, 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 degrees in orientation with respect to EpsJ; one loop of EpsJ is shifted by approximately 5A due to interactions with the nanobody; and a second loop of EpsJ underwent a major change of 17A without contacts with the nanobody. Clearly, nanobodies accelerate dramatically the crystallization of recalcitrant protein complexes and can reveal conformational flexibility not observed before.

DOI10.1016/j.jsb.2008.11.008
Alternate JournalJ. Struct. Biol.
PubMed ID19118632
Grant List5 T32 GM008268-19 / GM / NIGMS NIH HHS / United States
AI34501 / AI / NIAID NIH HHS / United States
/ / Howard Hughes Medical Institute / United States