Karyopherin-Centric Control of Nuclear Pores Based on Molecular Occupancy and Kinetic Analysis of Multivalent Binding with FG Nucleoporins

• Published in: Biophysical journal Vol. 106; no. 8; pp. 1751 - 1762
• Main Authors: Kapinos, Larisa E., Schoch, Rafael L., Wagner, Raphael S., Schleicher, Kai D., Lim, Roderick Y.H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2014; Biophysical Society; The Biophysical Society
• Subjects: Animals; beta Karyopherins - metabolism; Biochemistry; Glycine - analogs & derivatives; Glycine - metabolism; Humans; Kinetics; Models, Molecular; Molecules; Nuclear Pore - metabolism; Nuclear Pore Complex Proteins - chemistry; Nuclear Pore Complex Proteins - metabolism; Protein Binding; Protein Structure, Tertiary; Proteins; Proteins and Nucleic Acids; Surface Plasmon Resonance; Xenopus laevis
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2014.02.021

Intrinsically disordered Phe-Gly nucleoporins (FG Nups) within nuclear pore complexes exert multivalent interactions with transport receptors (Karyopherins (Kaps)) that orchestrate nucleocytoplasmic transport. Current FG-centric views reason that selective Kap translocation is promoted by alterations in the barrier-like FG Nup conformations. However, the strong binding of Kaps with the FG Nups due to avidity contradicts rapid Kap translocation in vivo. Here, using surface plasmon resonance, we innovate a means to correlate in situ mechanistic (molecular occupancy and conformational changes) with equilibrium (binding affinity) and kinetic (multivalent binding kinetics) aspects of Karyopherinβ1 (Kapβ1) binding to four different FG Nups. A general feature of the FxFG domains of Nup214, Nup62, and Nup153 is their capacity to extend and accommodate large numbers of Kapβ1 molecules at physiological Kapβ1 concentrations. A notable exception is the GLFG domain of Nup98, which forms a partially penetrable cohesive layer. Interestingly, we find that a slowly exchanging Kapβ1 phase forms an integral constituent within the FG Nups that coexists with a fast phase, which dominates transport kinetics due to limited binding with the pre-occupied FG Nups at physiological Kapβ1 concentrations. Altogether, our data reveal an emergent Kap-centric barrier mechanism that may underlie mechanistic and kinetic control in the nuclear pore complex.