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

• Published in: Nature communications Vol. 11; no. 1; p. 6179
• Main Authors: Nordeen, Sarah A, Andersen, Kasper R, Knockenhauer, Kevin E, Ingram, Jessica R, Ploegh, Hidde L, Schwartz, Thomas U
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 02.12.2020; Nature Publishing Group UK; Nature Portfolio
• Subjects: Accessibility; Amino Acid Sequence; Animals; Antibody Affinity; Antibody Specificity; Assembly; BASIC BIOLOGICAL SCIENCES; Binding Sites; Camelids, New World; Cloning, Molecular; Constituents; Crystallography, X-Ray; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Kinetics; Models, Molecular; Nanobodies; Nuclear Pore - chemistry; Nuclear Pore - metabolism; Nuclear Pore - ultrastructure; nuclear pore complex; Nuclear Pore Complex Proteins - chemistry; Nuclear Pore Complex Proteins - genetics; Nuclear Pore Complex Proteins - metabolism; Nucleoporins; Peptide Library; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; protein design; Protein Interaction Domains and Motifs; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae - ultrastructure; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Scaffolding; Sequence Alignment; Sequence Homology, Amino Acid; Single-Domain Antibodies - chemistry; Single-Domain Antibodies - genetics; Single-Domain Antibodies - isolation & purification; Single-Domain Antibodies - metabolism; Toolkits; x-ray crystallography; Yeast; Yeasts
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-19884-6

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.