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. 2015 Dec 3;4:e11349. doi: 10.7554/eLife.11349

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© 2015, Pleiner et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 8. — (a) Crosslinking of two different anti-Nup93 nanobodies (TP179 and TP324) to Nup93 using amine-to-amine ('Bis-NHS'; BS3; 11.4 Å linker length) or thiol-to-amine ('Mal-NHS'; BMPS; 5.9 Å linker length) crosslinking reagents. The combination of the very short Mal-NHS crosslinker with an engineered cysteine close to the antigen-binding loops provided for both nanobodies by far the highest yield of crosslinked nanobody•Nup93 adduct. (b) List of identified crosslinked peptides involving Nup93 lysines and Cys-TP179 or Cys-TP324. The crosslinked amino acids are highlighted in red (see also Figure 8—figure supplement 1). (c) Crosslinked lysines of Nup93 to the N-terminal cysteine on anti-Nup93 nanobodies TP179 (red) or TP324 (blue) are depicted on a structural model of Nup93^168-end generated by I-TASSER (Zhang, 2008). Based on the orthologous yeast crystal structures (Jeudy and Schwartz, 2007; Schrader et al., 2008), Nup93 is predicted to form a similar J-shaped structure (color gradient: NT = N-terminus in blue to CT = C-terminus in orange). Whereas TP179 binds to the central portion, TP324 binds to the C-terminus of Nup93.

DOI: http://dx.doi.org/10.7554/eLife.11349.013

Figure 8—figure supplement 1. — (a) Crosslinking of two different anti-Nup93 nanobodies (TP179 and TP324) to Nup93 using amine-to-amine ('Bis-NHS'; BS3; 11.4 Å linker length) or thiol-to-amine ('Mal-NHS'; BMPS; 5.9 Å linker length) crosslinking reagents. The combination of the very short Mal-NHS crosslinker with an engineered cysteine close to the antigen-binding loops provided for both nanobodies by far the highest yield of crosslinked nanobody•Nup93 adduct. (b) List of identified crosslinked peptides involving Nup93 lysines and Cys-TP179 or Cys-TP324. The crosslinked amino acids are highlighted in red (see also Figure 8—figure supplement 1). (c) Crosslinked lysines of Nup93 to the N-terminal cysteine on anti-Nup93 nanobodies TP179 (red) or TP324 (blue) are depicted on a structural model of Nup93^168-end generated by I-TASSER (Zhang, 2008). Based on the orthologous yeast crystal structures (Jeudy and Schwartz, 2007; Schrader et al., 2008), Nup93 is predicted to form a similar J-shaped structure (color gradient: NT = N-terminus in blue to CT = C-terminus in orange). Whereas TP179 binds to the central portion, TP324 binds to the C-terminus of Nup93.

DOI: http://dx.doi.org/10.7554/eLife.11349.013