Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Abstract

Nanobodies (Nbs) are popular and versatile tools for structural biology because they have a compact single immunoglobulin domain organization. Nbs bind their target proteins with high affinities while reducing their conformational heterogeneity, and they stabilize multi-protein complexes. Here we demonstrate that engineered Nbs can also help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interface. We have developed and characterised novel constructs, termed megabodies, by grafting Nbs into selected protein scaffolds to increase their molecular weight while retaining the full antigen binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we used a megabody to solve the 2.5 Å resolution cryo-EM structure of a membrane protein that suffers from severe preferential orientation, the human GABA A b3 homopentameric receptor bound to its small-molecule agonist histamine.

Structure, mechanism, and inhibition of Hedgehog acyltransferase

“…To structurally and functionally characterize HHAT, we raised camelid antibodies (nanobodies [NBs]) against full-length human HHAT and isolated two NBs (NB169 and NB177) that bound to HHAT expressed in cells and in biolayer interferometry using purified HHAT with low nanomolar affinity ( Figure S1 ). To overcome the technical hurdles of size and/or preferential orientation problems for cryo-EM structure analysis, we inserted the nanobodies into the E. coli K12 glucosidase YgjK scaffold resulting in ∼100 kDa megabodies (MBs; Uchański et al., 2021 ) (MB169 and MB177). We reconstituted the complex of human HHAT (incubated with nhPalm-CoA) and MB177 in detergent micelles and determined the cryo-EM complex structure to a 2.69-Å resolution using a localized refinement strategy ( Figure S2 ).…”

“…cDNA derived from Llama ( Lama glama ) serum was cloned into the pMESy4 (GenBank KF415192 ) phage display and bacterial expression vector. Megabody-expressing vectors were constructed as described in Uchański et al. (2021) via insertion of nanobody-encoding DNA into pMESP23E2 (GenBank: MT338521.1), which contains a circular permutant of Escherichia coli K12 glucosidase YgjK (UniprotKB: P42592 ; Gene ID: 947596) inserted into the first β-turn of NB169 or NB177.…”

Membrane Protein Stabilization Strategies for Structural and Functional Studies

“…On the other hand, adding nanobodies onto particular protein scaffolds results in increased molecular weight nanobody derivatives, named megabodies. These molecules have been used, among other applications, to obtain 3D reconstructions of membrane proteins that are too small to allow accurate particle alignment by Cryo-EM [121]. Nevertheless, the most straightforward application of nanobodies in structural biology is associated with the improvement of crystal diffraction quality [101,115].…”

Structure Determination of Inactive-State GPCRs with a Universal Nanobody

“…In the case of NTSR1/Nb6, vitrifying the sample in a very thin buffer layer on holey gold grids 20 was necessary, not only for high resolution but also to achieve proper alignment of particle projections. To assess an alternative approach, we used Nb6 to develop a megabody, termed 'Mb6', a fusion of a nanobody and a scaffold protein of either 45 or 86 kDa that can serve as a larger fiducial marker, based on the work of Uchański et al 6 . In that work, the Mb designs employed for GABAA and WbaP produced cryo-EM maps that could not resolve well past the nanobody portion, presumably due to flexibility.…”