Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

Abstract

Nanobodies are the recombinant antigen-recognizing domains of the minimalistic heavy chain-only antibodies produced by camels and llamas. Nanobodies can be easily generated, effectively optimized, and variously derivatized with standard molecular biology protocols. These properties have triggered the recent explosion in the nanobody use in basic and clinical research. This review focuses on the emerging use of nanobodies for understanding and monitoring protein dynamics on the scales ranging from isolated protein domains to live cells, from nanoseconds to hours. The small size and high solubility make nanobodies uniquely suited for studying protein dynamics by NMR. The ability to produce conformation-sensitive nanobodies in cells enables studies that link structural dynamics of a target protein to its cellular behavior. The link between in vitro and in-cell dynamics, afforded by nanobodies, brings the analysis of such important events as receptor signaling, membrane protein trafficking, and protein interactions to the next level of resolution.

Keywords: X-ray crystallography; nanobody; nuclear magnetic resonance (NMR); protein domain; protein dynamic; protein dynamics; protein engineering; protein structure; single-domain antibody (sdAb, nanobody).

FIGURE 1.

Domain structure of IgG antibodies (A) and heavy chain only camelid antibodies (B). The isolated variable domain of the latter is called nanobody.

FIGURE 2.

Schematic representation of the nanobody generation process, starting with the immunization of a camelid. PBMC, peripheral blood mononuclear cells; Ni-NTA, nickel-nitrilotriacetic acid; Nb, nanobody; IMAC, immobilized metal affinity chromatography; SEC, size exclusion chromatography.

FIGURE 3.

Structure of a nanobody (orange) in complex with lysozyme (green). CDR loops 1–3 are shown in light blue, navy, and red respectively. H-bonds between the nanobody and lysozyme are shown as blue bars. Note that out of nine intermolecular H-bonds, eight involve CDR3.

FIGURE 4.

Conformation and dynamics of metal-binding domains of ATP7B revealed by the nanobodies. A, a homology-based model of ATP7B structure (80) based on the x-ray structure of a copper-transporting ATPase CopA from Legionella pneumophila (81). A, N, and P denote the respective domains. Green ovals (MBDs) are the metal-binding domains of ATP7B absent in the CopA structure. B, a model of the chain of six MBD of ATP7B generated from the known MBD structures (red-yellow and cyan) (82–84). Nanobody bound to MBD3 is shown in orange. C, free MBD1–6 (left) and MBD1–6 with the 2R50 nanobody (orange) bound to MBD3 (right). Correlation times (τ_c), which characterize the rate of molecular tumbling, are shown next to each MBD. Changes in τ_c caused by the nanobody are color-coded as shown below. D, the difference between the transverse relaxation rates R₂ in the absence and presence of the 2R50 or 1R1 nanobody is plotted as a function of the residue sequence number. The locations of individual MBDs in the primary protein sequence are shown. Negative difference (green→blue) indicates a faster relaxation (slower tumbling) with nanobody bound. Conversely, positive difference (green→red) corresponds to a slower relaxation in the presence of nanobody and thus faster tumbling.