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. While 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.

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

“…Finally, addition of any enzymatic domain to the small tag binders would allow the specific modification of the tagged POI, as it was elegantly shown with a minimal Rho kinase domain fused to the GFP nanobody to phosphorylate a GFP-tagged protein in Drosophila melanogaster ( Roubinet et al, 2017 ) or with a proximity-directed O-GlcNAcetylation linking the O-GlcNAc transferase activity to the GFP or EPEA nanobody in cell culture ( Ramirez et al, 2020 ). Several recent reviews have highlighted the versatility of the nanobodies for numerous applications both in clinical and biological research ( Beghein and Gettemans, 2017 ; Cheloha et al, 2020 ; Ingram et al, 2018 ; Muyldermans, 2020 ; Schumacher et al, 2018 ; Yang and Shah, 2020 ). The various functionalization strategies can be extended to these small tag binders.…”

Generation and characterization of a laforin nanobody inhibitor

“…This work has been hampered due to the molecular dynamics of wildtype and mutant enzymes. Nanobodies can be employed to limit protein dynamics and there have been impressive successes in this area 52 . Crystallizing laforin with the nanobodies could capture conformations that are important for its biological function.…”

“…Glycogen contains 1 phosphate per 1,000-10,000 glucose residues and amylopectin contains 1 phosphate per ~300 glucose residues 5,42,[48][49][50][51] . Nanobodies are a rapidly growing technology that are being utilized in novel ways to refine a wide variety of traditional techniques such as crystallization chaperoning, affinity purification, immunoprecipitation, superresolution microscopy, confocal microscopy, flow cytometry, cell delivery, radiolabeling, and modulating protein function and interactions in cells 52,53 . An immediate opportunity to utilize the laforin nanobodies is with respect to modulating laforin's phosphatase activity, glycogen binding, and interactions.…”

A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure

“…Regardless of this difference, both have very high binding affinities (Table 1). VHH-SAN10 and 11 add to a growing list of nanobodies that bind to short epitopes that function outside of a folded domain [41][42][43][44] .…”