A general protocol for the generation of Nanobodies for structural biology

Abstract

There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo-matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6-12 months.

Figure 1

Workflow for generating conformational Nanobodies for structural biology.

Figure 2

Native gel analysis of Nanobodies interacting with the OB fold of the A1 protein of the editosome of the sleeping sickness parasite Trypanosoma brucei . Nanobodies Nb7 and Nb8 were generated using this protocol. The target protein was incubated with Nb7 or Nb8 for 30 min at 4 °C in 20 mM Tris (pH 7.5), 1 mM DTT and 300 mM NaCl. Complex formation of target and Nanobody was analyzed on a 4-15% native gel at 110 V for 1 h and stained by Coomassie Blue. The positions of the target protein alone (lane 1), the target•Nb7 complex (lane 3), the Nanobody Nb8 alone (lane 4), and the target•Nb8 complex (lane 5) are indicated with symbols above the bands. Due to its high isoelectric point, Nb7 is too positively charged to run into the native gel (lane 2).

Figure 3

Nb80 is a structural mimic of GαS and stabilizes the active-state conformation of β₂AR. Nb80 (red) and GαS (green) bind the same intracellular cavity of the agonist bound β₂ adrenoreceptor in the β₂AR•Nb80 complex (PDB3P0G; β₂AR in orange, Nb80 in red) and the β₂AR•Gs complex (PDB3SN6; β₂AR in gray, GαS in green, Gβ in salmon and Gγ in blue).

Figure 4

Structure of the β₂AR•Gs complex solved by Nanobody-enabled X-ray crystallography. Surface representation of the active state ternary complex composed of agonist-occupied monomeric β₂AR (grey) and nucleotide-free Gs heterotrimer. Nb35 (Red, cartoon representation) binds at the interface of the Gα (green) and Gβ (salmon) subunits. Gγ is represented in blue.