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

Abstract

Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe

“…However, the increased frequency of ring clusters in nup132Δ cells may provide insights into the mechanism driving their formation. Nup132 is a structural Nup that facilitates interactions between the structural scaffold of the NPC with lipids through its N-terminal ALPS motif (115,116). Deletion of Nup132 may alter the interactions between NPCs and specific lipid species present in the NE, making nup132Δ cells especially sensitive to changes in lipid composition that may occur due to nutrient availability during growth on plates.…”

VHH Structural Modelling Approaches: A Critical Review

“…This particular V H H was used to understand the structural basis for the induction of a spike trimer [ 144 ]. Noorden and co-workers built a complex of a V H H and SAN2 protein and provided further comprehension of a nuclear pore complex structure [ 145 ]. Thanongsaksrikul and co-workers developed libraries of VHs and V H Hs against botulism.…”

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralizing SARS-CoV-2 nanobodies

“…Their small size and single-gene nature allow for easy cloning, modification, and functionalization and also permit better tissue penetration and, for imaging purposes, closer proximity of fluorophores or radioisotopes to the antigen. As a result, nanobodies have applications in cell biology ( 2 ), structural biology ( 3 ), cancer research ( 4 ), and immunology ( 5 ). In addition, nanobodies are ideal neutralizing molecules or perturbants of viruses.…”