FC0098
Nup153  -  Importinβ

Biological function
Nucleoporins establish a permeability barrier to selectively allow entry of nuclear transport receptors (NTRs).

Domain organization/sequence features
The phenylalanine-glycine rich (FG-rich) region of Nups binds NTRs with near diffusion limited rates.

Structural evidence
¹D_N-NH and ¹D_Ca-Ha residual dipolar couplings and SAXS shows negligible secondary structure for isolated Nup153FG^PxFG in solution. smFRET demonstrates that global structure and dynamics of the Nup153FG^PxFG
 are retained upon interaction with importinβ. Similarly, ¹⁵N relaxation rates show that global structure and flexibility of Nup153FG^PxFG are unaffected by importinβ binding, rather local and transient changes in these properties are observed. A fast exchange (< 10 ms) between the bound and unbound form of Nup153FG^PxFG was found, FG- specific K_d,individual values are in the mM range. Comparison of ¹³C backbone chemical shifts measured in the free and NTF2-bound forms of Nup153FG^PxFG demonstrates that the protein backbone remains flexible upon interaction, sampling effectively the same conformational equilibrium in the free and bound state.

Biochemical evidence
Mutation of phenylalanines of Nup153FG^PxFG (except F1374) by alanine show a strongly reduced peak broadening upon titration with importinβ. The estimated K_d,individual values are 7.3 mM and 0.8 mM using the mutant and the wild-type construct, respectively. Fluorescence anisotropy measurements indicate an ultrafast k_on,ultrafast = 1.5x10⁹ M^-1s^-1.

Structure/Mechanism
The fast binding rates mainly originate mostly from the desolvation effect of phenylalanines, as association remains very fast with k_{on,elect off} of 2.9x10⁸ M^-1s^-1 even under electrostatic shielding. smFRET, NMR and simulations consistently demonstrate that FG-Nup maintains its conformational ensemble upon binding to the NTR. NMR studies suggest that individual FG-motifs bind independently of each other, as the ¹⁵N R₂ rates are similar to the values of the unbound Nup between the FG-repeats. Multivalency of the system however increases association rates by several orders of magnitude. Many individual low-affinity motifs paired with a binding mode that requires relatively little energy or time investment for the Nup to transit between free and bound conformations.

Mechanism category
tethering

Significance
Fuzziness in a multivalent complex enables ultrafast association with the partner via many weak-affinity interactions. Binding of NTRs leaves the conformational ensemble and dynamics of FG-Nups largely unaffected that provides a rationale for the fast, yet specific, nuclear transport.

Submitted by
Edward Lemke   lemke@embl.de