Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus.
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.
Lam
Anita Y
AY
Department of Biochemistry, Biomolecular Structure Center, University of Washington, 1959 Pacific Ave. NE, HSC K-428, Seattle, WA 98195, USA.|Biomolecular Structure & Design Program, Biomolecular Structure Center, University of Washington, Seattle, WA 98195, USA.
Pardon
Els
E
Department of Molecular and Cellular Interactions, VIB, B-1050 Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium.
Korotkov
Konstantin V
KV
Department of Biochemistry, Biomolecular Structure Center, University of Washington, 1959 Pacific Ave. NE, HSC K-428, Seattle, WA 98195, USA.
Hol
Wim G J
WGJ
Department of Biochemistry, Biomolecular Structure Center, University of Washington, 1959 Pacific Ave. NE, HSC K-428, Seattle, WA 98195, USA.|Biomolecular Structure & Design Program, Biomolecular Structure Center, University of Washington, Seattle, WA 98195, USA.
Steyaert
Jan
J
Department of Molecular and Cellular Interactions, VIB, B-1050 Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000737:chemistry; D001666:Binding Sites, Antibody; D002161:Camelids, New World / Q000276:immunology; D022801:Complementarity Determining Regions / Q000737:chemistry; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000737:chemistry; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D041261:Vibrio vulnificus / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D022801:Complementarity Determining Regions; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins