Nuclear transport receptor binding avidity triggers a self-healing collapse transition in FG-nucleoporin molecular brushes

Abstract

Conformational changes at supramolecular interfaces are fundamentally coupled to binding activity, yet it remains a challenge to probe this relationship directly. Within the nuclear pore complex, this underlies how transport receptors known as karyopherins proceed through a tethered layer of intrinsically disordered nucleoporin domains containing Phe-Gly (FG)-rich repeats (FG domains) that otherwise hinder passive transport. Here, we use nonspecific proteins (i.e., BSA) as innate molecular probes to explore FG domain conformational changes by surface plasmon resonance. This mathematically diminishes the surface plasmon resonance refractive index constraint, thereby providing the means to acquire and correlate height changes in a surface-tethered FG domain layer to Kap binding affinities in situ with respect to their relative spatial arrangements. Stepwise measurements show that FG domain collapse is caused by karyopherin β1 (Kapβ1) binding at low concentrations, but this gradually transitions into a reextension at higher Kapβ1 concentrations. This ability to self-heal is intimately coupled to Kapβ1-FG binding avidity that promotes the maximal incorporation of Kapβ1 into the FG domain layer. Further increasing Kapβ1 to physiological concentrations leads to a "pileup" of Kapβ1 molecules that bind weakly to unoccupied FG repeats at the top of the layer. Therefore, binding avidity does not hinder fast transport per se. Revealing the biophysical basis underlying the form-function relationship of Kapβ1-FG domain behavior results in a convergent picture in which transport and mechanistic aspects of nuclear pore complex functionality are reconciled.biointerface | molecular crowding | multivalent binding | nucleocytoplasmic transport | polymer brush I ntrinsically disordered proteins (IDPs) that adorn the surfaces of biomolecular structures are thought to confer a host of unique functionalities not found in structured proteins (1). However, unlike their free-floating counterparts in solution (2), the properties of such surface-tethered IDPs can be particularly challenging to evaluate because of their inherent flexibility and conformational susceptibility to local interfacial constraints (3). Herein lies the crux of the nuclear pore complex (NPC) problem, in which in vitro efforts (4-11) to rationalize the collective formfunction characteristics of the intrinsically disordered Phe-Gly (FG) domains (12) typically neglect the uncertainty regarding their numbers [approximately 200 divided amongst 11 different FG-bearing nucleoporins (Nups) (13)], their locations within the central NPC channel, and corresponding distances between neighboring anchoring sites (14). As the key components of the NPC barrier mechanism, the manner by which the FG domains impede nonspecific molecules (greater than 40 kDa) whilst granting karyopherins (Kaps) and their cargoes access between the nucleus and the cytoplasm (15-17) is likely to be influenced by such physical constraints. Accordingly, it remains unaccounted for how these contextual detail...

Stimuli-sensitive intrinsically disordered protein brushes

“…IDP sequences can be optimized for specific applications through directed-evolution approaches and synthesized to include bio-orthogonal chemical moieties via the use of unnatural amino acids. Indeed, IDPs based on nucleoporin proteins have recently been incorporated into synthetic gated pores 47 and surface-grafted layers 48 , supporting the idea that IDPs can be manipulated in ways that are analogous to synthetic polymers. Together with these efforts, our work hints at the value of exploring IDPs as a new class of 'smart' biomaterial building blocks.…”

How to operate a nuclear pore complex by Kap-centric control

“…21 Stepwise increases in Kapb1 concentrations then led to a gradual re-extension of the FG layer. 26,44,48 This socalled "self-healing" can be attributed to an increase in population (and volume) of bound Kapb1 within the FG layers, as predicted by Zilman theory. 50,51 It should be noted that this was true for Nup153, Nup214, and Nup62 and its yeast ortholog Nsp1, with the exception of Nup98, 26 which exhibited pronounced cohesion based on its poor extensibility and low capacity for incorporating Kapb1.…”

“…26,44,48 This indicates that the occupancy of bound Kapb1 modulates subsequent kinetic behavior in a differential manner, being attributed to how many available FG-repeats each incoming Kapb1 molecule can access and bind to. Hence, fast transport emerges from a reduction of avidity due to a saturation of the FG Nup layer with pre-bound Kapb1.…”

“…This "peeling open" of the pore was achieved using a surface plasmon resonance (SPR) technique that we developed to correlate in situ equilibrium and kinetic aspects of Kapb1-FG Nup binding to changes in FG layer conformation and Kapb1 occupancy. 48 Briefly, this used BSA molecules as innate molecular probes that could "feel" the physical barrier properties of a surfacetethered molecular layer 49 by not binding the FG Nups.…”

Crosslink-induced shrinkage of grafted Gaussian chains

“…For example, the thickness elasticity of the red cell membrane has been related to the entropy of spectrin loops grafted on the lipid bilayer [4]. Cross-link induced grafted polymer collapse has been proposed as part of the mechanism for the selective gating in the nuclear pore complex which regulates cargo transport between the cytoplasm and the nucleus in eukariotic cells [5,6]. If the grafting density is low, collective stretching effects become negligible and the conformations of the chains are determined by their bending stiffness and the boundary conditions imposed by the substrate.…”