Title | Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus. |
Publication Type | Journal Article |
Year of Publication | 2009 |
Authors | Lam, A. Y., E. Pardon, K. V. Korotkov, W. G. J. Hol, and J. Steyaert |
Journal | J Struct Biol |
Volume | 166 |
Issue | 1 |
Pagination | 8-15 |
Date Published | 2009 Apr |
ISSN | 1095-8657 |
Keywords | Amino 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. |
DOI | 10.1016/j.jsb.2008.11.008 |
Alternate Journal | J. Struct. Biol. |
PubMed ID | 19118632 |
Grant List | 5 T32 GM008268-19 / GM / NIGMS NIH HHS / United States AI34501 / AI / NIAID NIH HHS / United States / / Howard Hughes Medical Institute / United States |
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