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The nuclear pore permeability barrier – physical concepts and a biosynthetic approach to understand and exploit the unique selectivity of a natural macromolecular sieve

Final Report Summary - NUCLEAR PORE (The nuclear pore permeability barrier – physical concepts and a biosynthetic approach to understand and exploit the unique selectivity of a natural macromolecular sieve)

Nuclear pore complexes (NPCs) are the principal gate of entry/exit of macromolecules into and out of the nucleus of living cells. They exhibit a unique selectivity in transport, with respect to size and molecular species: very small molecules can diffuse efficiently through the pore, while larger objects are delayed/blocked unless they are bound to specialized proteins, so called nuclear transport receptors (NTRs). The permeability barrier and its selectivity arise from an assembly of protein domains that are rich in phenylalanine-glycine repeats (FG repeat domains). These domains are natively unfolded and grafted at high density to the NPC channel walls. NTRs can interact with the FG repeat domains, thereby facilitating translocation of NTR-bound cargo.

The supramolecular organization of the nuclear pore permeability barrier and the mechanism behind selective transport remain insufficiently understood. The goal of this project was to understand the relation between the organizational and dynamic features of FG-nucleoporin assemblies, their physicochemical properties, and the resulting biological functions. We quantitatively studied these relations on the supramolecular level, a level that – for this type of assemblies – is hardly accessible with conventional biological and biophysical approaches. For this purpose we combined quartz crystal microbalance (QCM-D) and spectroscopic ellipsometry (SE) in situ to quantitatively analyze the binding of NTRs to films of end-grafted FG domains as a bottom-up nanoscale model system of the permeability barrier. For our measurements we used both naturally occurring and artificially designed FG repeat domains.

In a series of experiments we titrated FG repeat domain films with increasing concentrations of selected NTRs. The films were made of naturally occurring and of artificially designed FG repeat domains. We combined data from QCM-D and SE measurements to obtain thickness changes and NTR partition coefficients for various NTR concentrations in solution. We were able to demonstrate that the binding of NTRs to FG repeat domain films cannot be described by commonly established binding models. We elucidated possible reasons for this non-ideal behavior (e.g. the influence of molecular crowding) and discussed arising effects for physico-chemical parameters (e.g. an apparent concentration dependence of the equilibrium constant for NTR binding) and resulting implications for nuclear transport. Furthermore, simulations performed by our collaborators validated our data and allowed us to obtain information that is not easily accessible with experimental methods (e.g. the spatial distribution of NTRs in FG repeat domain films at different NTR concentrations).

Naturally occurring FG repeat domains show a large variability in amino acid sequence and their FG repeats are often surrounded by different amino acids, even within the same FG repeat domain. Also the spacer sequences that separate two FG motifs are normally of different length and of different amino acid composition. This irregularity makes it difficult to quantitatively understand the interactions between FG repeat domains or between FG repeat domains and NTRs. In our experiments we used artificially created FG repeat domains with specific amino acid mutations and characterized the nanoscale organization (thickness, viscoelastic properties) and the functionality (avidity to NTRs) of the resulting FG repeat domain films. We showed that changing the amino acids in proximity of the FG motifs or changing the electrostatic charge of FG repeat domains considerably changed their avidity to NTRs.

The results of our project advance the mechanistic understanding of nuclear transport. Although this research field is very active, to date, there is no conclusive theory available describing the nanoscale organization of the nuclear permeability barrier and explaining its mechanism of function. Several distinct models have been proposed and partially contradict each other. The results of our titration studies and the concomitant computer simulations allow us to rule out certain aspects of these theories. This way, our study has a strong impact in fundamental biology and results in a better understanding of the organization and function of the nuclear pore permeability barrier. In addition, our results involving the artificial FG repeat domains are potentially of major medical relevance, because some viruses overcome the nuclear barrier by binding to the FG motifs in a similar way as NTR-mediated transport. Thus, studying viral binding to specifically mutated FG repeat domains could open, at long term, a new pathway for targeting viral infections.