Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargo. A particular class of Nup (FG Nups) has intrinsically disordered regions containing phenylalanyl-glycyl rich repeats (FG Nups) that interact with TFs to ensure their selective translocation across the NPC. How selective transport is achieved at the molecular level remains unclear, because key atomic-scale details of FG Nup behavior and TF-FG Nup interactions have been missing. We have combined in vitro transport systems with NMR spectroscopy to determine the atomic-scale behavior of all residues in the FG repeats. These experiments reveal that while blocking the passage of other macromolecules. underlying mechanism of rapid, selective transport . We show that cellular milieux stabilize the FG repeats in a fully disordered state, forming a dynamic tethered fluid, and that the combined effect of many frequent and transient interactions between multiple phenylalanine binding sites on the TFs and the FG repeats provides a robust, tunable and selective transport system. Taken together, we have shown that we can determine the dynamic atomic scale behavior of key aspects of the structure and dynamics of selected FG repeat regions in the presence of their normal cellular environment and typical TFs, definitively demonstrating the tremendous and broad-reaching potential of these approaches. Our objectives now are to apply these orthogonal and highly innovative approaches, primarily using NMR, to obtain complete atomic scale dynamic pictures of the functional roles of FG repeat regions at three levels: (i) underlying polymer property and environmental influences; (ii) interactions with TFs and other macromolecules, including understanding how non-specific macromolecules are excluded; and (iii) the influence of specific topology and ensemble properties of the transporting NPC. The impact of this research will be a substantial increase in our knowledge of transport through the NPC, novel methods for probing IDPs of pathological significance and their interactions in cells, and direct linkage of atomic scale information of FG repeat properties to transport phenomena. In addition, it is likely that new approaches to describing the unrecognized properties and functions of unfoldable intrinsically disordered proteins will be produced and impact in other areas, e.g. amyloidosis diseases. This proposal addresses a fundamental knowledge gap in the area of selective diffusion in biology, which may permit rational therapeutic approaches and molecular design related to the NPC for e.g. neoplasms, and viral invasion.

Public Health Relevance

Natural variations or viral usurpation of the parts of the pores permitting materials to go in and out of the nucleus of the cell cause susceptibility to viruses, heart disorders, and selective growth of cancers. New approaches to characterizing these parts will permit better understanding of the causes of the diseases and potentially point to therapeutic approaches in these and related diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM117212-05
Application #
9623958
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter
Project Start
2016-03-01
Project End
2020-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
5
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
DUNS #
081266487
City
Bronx
State
NY
Country
United States
Zip Code
10461
Sparks, Samuel; Temel, Deniz B; Rout, Michael P et al. (2018) Deciphering the ""Fuzzy"" Interaction of FG Nucleoporins and Transport Factors Using Small-Angle Neutron Scattering. Structure 26:477-484.e4
Hayama, Ryo; Sparks, Samuel; Hecht, Lee M et al. (2018) Thermodynamic characterization of the multivalent interactions underlying rapid and selective translocation through the nuclear pore complex. J Biol Chem 293:4555-4563
Upla, Paula; Kim, Seung Joong; Sampathkumar, Parthasarathy et al. (2017) Molecular Architecture of the Major Membrane Ring Component of the Nuclear Pore Complex. Structure 25:434-445