Structural and biochemical characterization of nuclear pore complex structural scaffold sub-complexes

Abstract

The nuclear pore complex (NPC) is a large, modular protein assembly that regulates nucleocytoplasmic transport in all eukaryotes. The ~60-120 MDa NPC is a modular assembly of multiple copies of ~30 distinct proteins that are arranged into biochemically distinct sub-complexes. We believe that the structural characterization of the NPC is essential for understanding its transport mechanisms and various pathologies and human diseases associated with deletions or mutations of constituents. To obtain detailed structural information of the NPC, techniques that span several resolution ranges are necessary due to its large size and complexity. For instance, recent progress in the structural characterization of the overall architecture of the NPC by cryo-electron tomography(cryo-ET) to ~23Å resolution has revealed its size, shape, and course arrangement, but lacks distinguishable protein-protein boundaries and secondary structural details.

Although the entire NPC is not amenable to high resolution X-ray crystallography, we complement the cryo-ET reconstructions with a divide and conquer approach by obtaining high resolution X-ray crystal structures of individual subcomplexes. By taking advantage of the modular nature of the NPC, we can dock subcomplexes into the cryo-ET reconstructions to identify their location within the NPC. This composite structure will bridge the meso resolution cryo-ET reconstructions of the entire NPC and the incomplete but high resolution X-ray crystal structure of individual subcomplexes. As a first step towards understanding the detailed organization of the NPC, our goal is to solve the high resolution structures of the two principal structural scaffold subcomplexes, the Y and Nic96 complexes. In this study, we present the high resolution composite X-ray crystal structure of the Y complex.

Docking the composite model into previously solved random conical tilt(RCT) and tomographic reconstructions of negatively stained samples of the Y complex shows overall consistency between the three methods, yet we highlight important structural differences that constrain possible arrangements of multiple Y complexes within the NPC. By docking the composite model into the cryo-ET reconstructions of the entire NPC, we propose an arrangement of multiple Y complexes that is consistent with our composite structure. In addition, progress on structurally characterizing the Nic96 complex will be presented. Preliminary results suggest that Nup1 92 and Nic96 form a flexible, yet semi-ordered interface. Future directions for characterizing the rest of the Nic96 complex, including current challenges and suggestions will be discussed.