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

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, that can be classified into three categories: 1. Stably associated scaffolding nups, 2. Peripheral nups, and 3. Phenylalanine-glycine (FG) repeat containing nups that form the permeability barrier of the NPC. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 complex. Working in S. cerevisiae to study the assembly of these two essential subcomplexes, we developed a suite of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. The nanobodies bind their targets specifically and with high affinity, albeit with varying kinetics. We mapped the epitope of eight members of the nanobody library via crystal structures of nup-nanobody co-complexes.

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, that can be classified into three categories: 1. Stably associated scaffolding nups, 2. Peripheral nups, and 3. Phenylalanine-glycine (FG) repeat containing nups that form the permeability barrier of the NPC. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 complex. Working in S. cerevisiae to study the assembly of these two essential subcomplexes, we developed a suite of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. The nanobodies bind their targets specifically and with high affinity, albeit with varying kinetics. We mapped the epitope of eight members of the nanobody library via crystal structures of nup-nanobody co-complexes.

In two cases, the nanobodies facilitated the crystallization of novel nup structures, namely the full-length Nup84-Nup133 [alpha]-helical domain structure and the Nup133 [beta]-propeller domain structure. Together these two structures completely characterize the S. cerevisiae Y complex molecular assembly. Further, the Nup133 [beta]-propeller domain contains a structurally conserved amphipathic lipid packing sensor (ALPS) motif thought to anchor the Y complex to the nuclear envelope, which we confirmed by liposome interaction studies. An additional nanobody facilitated the structure of Nic96 at an improved resolution, revealing previously missing helices. In addition to the utility of these nanobodies for in vitro characterization of NPC assemblies, we also show that expression of nanobody-fluorescent protein fusions reveals details of the NPC assembly in their native, in vivo environment, and possibly of NPC heterogeneity within the nuclear envelope.

Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.