Exploring cellular biochemistry with nanobodies

Abstract

Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain-only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.

Keywords: antibody engineering; cell signaling; protein chemistry; single-domain antibody (SdAb); synthetic biology.

Figure 1.

Structures of human and camelid Igs and fragments. Conventional human Igs (i.e. IgG) have been truncated to provide functional fragments (Fab and scFv) that contain variable regions from the light and heavy chains. In the case of the scFv, a linker is required to facilitate appropriate pairing of heavy- and light-chain variable regions. A subset of antibodies from camelids consists of only the heavy chains. Expression of the isolated variable region from heavy chain–only antibodies provides functional single-domain antibodies (VHHs/nanobodies).

Figure 2.

Recent examples of nanobody bioconjugation. A, enzymatic approaches, including OaAEP1 (a), formylglycine-generating enzyme (b), tubulin Butelase and tubulin tyrosine ligase need to be switched in the figure legend (Butelase is panel c, Tubulin tyrosine ligase is panel d) and sortase A (e). B, incorporation of unnatural amino acids by stop codon suppression. C, expressed protein ligation. See the Chemical and Enzymatic Functionalization section for a discussion of strengths and drawbacks of these approaches and associated references.

Figure 3.

Three examples of nanobodies used as crystallization chaperones. A, in the structure of nucleoporin Nup133 from S. cerevisiae, three nanobodies (shades of orange) generated the critical packing interface necessary to build up the crystal lattice (PDB code 6X04). B, in the structure of the nucleoporin complex of Nup107 and Nup133 from H. sapiens two different nanobodies that bind the Nup107 moiety in separate locations were co-crystallized (PDB code 6X03). C, in the TorsinA-LULL1 complex structure, the nanobody recognizes both binding partners and binds neither TorsinA (white) nor LULL1 (gray) individually (PDB codes 5J1S and 5J1T).

Figure 4.

Nanobody-ligand conjugates to target a G protein–coupled receptor. Synthetic fragments of parathyroid hormone were site-specifically linked to nanobodies to provide conjugates (bottom right) with biological activity (EC₅₀) superior to the free ligand (bottom left). Structures are based on human parathyroid hormone receptor (gray) in complex with PTH (orange) (PDB code 6FJ3) and a generic VHH (blue) with complementarity-determining regions highlighted (red) (PDB code 3K1K). The binding of the nanobody to PTHR1 (bottom right) is shown in two possible orientations as the actual site of binding is unknown.

Figure 5.

Nanobodies as redirecting and sensing agents in live cells. A, use of GFP-binding nanobodies to redirect tagged proteins to subcellular locations or for degradation. B, use of a p53-binding nanobody to block HPV E6-mediated ubiquitination and degradation. C, use of orthogonal anti-NP nanobodies as biosensors coupled to a transcriptional output (214). The DNA-binding domain (DBD) and VP64 activation domain are separately fused to anti-NP nanobodies (VHHs). UAS, upstream activator sequence that binds DBD. Transcription of the reporter gene produces GFP. D, use of nanobodies as biosensors to detect active and inactive states of GPCRs.

Figure 6.

Application of nanobodies in synthetic biology. A, use of nanobodies as recognition elements in programmable synNotch constructs (225). Upon ligand binding, sometimes found on the surface of neighbor cells, synNotch receptors undergo a conformational change that promotes cleavage and release of an intracellularly linked transcription factor. B, nanobodies as recognition elements in bacterial constructs for sensing of extracellular ligands (226). The addition of caffeine causes clustering of extracellular single-domain antibodies and subsequent assembly of split DBDs and a transcriptional output. C, scheme for attachment of nanobodies to bacterial outer membranes or OMVs using SpyCatcher/SpyTag (229). An outer membrane protease, hemoglobin protease (gray), is linked to SpyTag (red). A SpyCatcher/nanobody fusion (black/gold) covalently labels the hemoglobin protease-SpyTag fusion.