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

Fig. 1

Anti-EpsI:EpsJ generated nanobodies and complex formation with EpsI:EpsJ. (A) Sequence alignment of nanobodies generated against V. vulnificus EpsI:EpsJ. The nanobodies that bound to EpsI:EpsJ according to gel-shift assays (Fig. 1B) are titled in red. While the numbering on top of the alignment corresponds to the continuous numbering present in the PDB file, the numbering on the bottom of the alignment corresponds to the standard IMGT numbering for antibodies and related proteins (Lefranc, 2005; Supplementary Figure S1). The conserved cysteines that form the intra-molecular disulfide bridge are highlighted in blue while the C-terminal hexahistidine tag is highlighted in yellow. Boxed segments of sequence compose the CDR regions with the green box as CDR1, purple box as CDR2, and red box as CDR3. Green stars denote residues that make contacts with EpsJ. Cyan and purple triangles reflect residues that make nanobody-nanobody contacts, with cyan as those of Chain C and purple of Chain F. (B) Ternary complex formation of N-terminal His₆ EpsI:EpsJ with nanobodies. A native PAGE gel of anti-EpsI:EpsJ nanobodies alone and with V. vulnificus EpsI:EpsJ. The majority of the nanobodies do not enter the gel due to the high pI's that these protein exhibit (~9). Only NbEpsIJ-13 and NbEpsIJ-19, with pI = 8.0, enter the gel and are seen in the nanobody control lanes. Ternary complex formation between EpsI:EpsJ and the nanobody (NbEpsIJ-11, NbEpsIJ-12, NbEpsIJ-17, NbEpsIJ-19) cause a dramatic band shift in the gel and are denoted by asterisks. The EpsI:EpsJ control is seen in the far right lane. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)

Fig. 2

The structure of V. vulnificus EpsI:EpsJ in complex with Nb11. (A) View of the unit cell with four EpsI:EpsJ:Nb11 ternary complexes. EpsI, light blue; EpsJ, blue; and Nb11, orange. The cartoon representation of the unit cell is seen on the left, and the surface representation is seen on the right. The chain names in the structure are next to their respective components of the unit cell. (B) General architecture and secondary structure elements of the EpsI:EpsJ:Nb11 ternary complex. EpsI, light blue; EpsJ, blue; and Nb11, orange. Nb11 is further colored by CDR, with CDR1 being green, CDR2 purple, and CDR3 red. Each α-helix and β-strand has been labeled according to its order in the protein. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)

Fig. 3

Comparison of EpsI:EpsJ heterodimers from two different structures. The EpsI:EpsJ:Nb11 structure superimposed onto the EpsI:EpsJ structure from Yanez et al. (2008b) by using only the EpsJ chains for calculating the superposition operation. The EpsI (cyan) chains differ in orientation by ~6°. Two loops differing in conformation, discussed in the text, are shown in brown in the EpsI:EpsJ:Nb11 structure and yellow in the EpsI:EpsJ structure from Yanez et al. (2008b). (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)

Fig. 4

The interacting surfaces of V. vulnificus EpsJ and Nb11. (A) EpsJ is shown in blue with the residues that interact with the Nb11 CDR regions color-coded. CDR1 interactions, green; CDR2 interactions, purple; CDR3 interactions, red. The orange surface area represents residues of EpsJ interacting with the Nb11 framework. As orange ribbons are depicted the main-chain loops of the three Nb11 CDR's and framework residues interacting with residues of EpsJ. (B) “Butterfly” representation of EpsJ:Nb11 complex. Left: EpsJ: spheres denoting residues that interact with Nb11. CDR1-interacting residues, green; CDR2-interacting residues; purple; CDR3-interacting residues, red; framework-interacting residues, orange. Right: Nb11: spheres denoting residues that interact with EpsJ. CDR1, green; CDR2, purple; CDR3, red; framework, orange. “PDB entry 3CFI residue numbering” is used; see upper line of Fig. 1A. The corresponding “IMGT numbering” is shown in the lower line of Fig. 1A. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)

Fig. 5

View of the “layers” of the heterotrimer in the crystal lattice. Crystal contacts between the nanobodies within the crystal allow the lattice to be composed of layers of EpsI, EpsJ, and Nb11 molecules. Nb11 in gold; EpsI in light blue; EpsJ dark blue. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)