The central transport channel (CTC) of nuclear pore complexes (NPCs) is made up of three nucleoporins Nup62, Nup58 and Nup54. In which manner and capacity, these nucleoporins form the CTC, is not yet clear. We explored the CTC Nups from various species and observed that distinct biochemical characteristics of CTC Nups are evolutionarily conserved. Moreover, comparative biochemical analysis of CTC complexes showed various stoichiometric combinations of Nup62, Nup54 and Nup58 coexisting together. We observed the conserved amino-terminal domain of mammalian Nup93 is crucial for the anchorage of CTC and its localization to NPCs. We could reconstitute and purify mammalian CTCÁNup93 quaternary complex by co-expressing full length or N-terminal domain of Nup93 along with CTC complex. Further, we characterized CTCÁNup93 complex using small angle X-ray scattering and electron microscopy that revealed a "V" shape of CTCÁNup93 complex. Overall, this study demonstrated for the first time evolutionarily conserved plasticity and stoichiometric diversity in CTC Nups. K E Y W O R D S central transport channel, nuclear pore complex, Nup93, small angle X-ray scattering, co-immunoprecipitation Parshuram J. Sonawane and Pravin S. Dewangan are lead co-authors.

Nuclear pore complex (NPC) is the largest multimeric protein assembly of the eukaryotic cell, which mediates the nucleocytoplasmic transport. The constituent proteins of this assembly (nucleoporins) are present in varying copy numbers to give a size from ~ 60 MDa (yeast) to 112 MDa (human) and share common ancestry with other membrane‐associated complexes such as COPI/COPII and thus share the same structural folds. However, the nucleoporins across species exhibit very low percentage sequence similarity and this reflects in their distinct secondary structure and domain organization. We employed thorough sequence and phylogenetic analysis guided from structure‐based alignments of all the nucleoporins from fungi to metazoans to understand the evolution of NPC. Through evolutionary pressure analysis on various nucleoporins, we deduced that these proteins are under differential selection pressure and hence the homologous interacting partners do not complement each other in the in vitro pull‐down assay. The super tree analysis of all nucleoporins taken together illustrates divergent evolution of nucleoporins and notably, the degree of divergence is more apparent in higher order organisms as compared to lower species. Overall, our results support the hypothesis that the protein–protein interactions in such large multimeric assemblies are species specific in nature and hence their structure and function should also be studied in an organism‐specific manner.

7 Motivation 8 Decrypting the interface residues of the protein complexes provide insight into the functions 9 of the proteins and hence the overall cellular machinery. Computational methods have been 3 1 RNA and proteins interact with each other. To understand the overall functioning of the cell, 3 2it is important to delineate the pairwise interactions of these basic units such as DNA-protein, 3 3

No abstract