Structural Mechanism of Nuclear Transport Mediated by Importin β and Flexible Amphiphilic Proteins

Abstract

Karyopherin β family proteins mediate the nuclear/cytoplasmic transport of various proteins through the nuclear pore complex (NPC), although they are substantially larger than the size limit of the NPC.To elucidate the molecular mechanism underlying this paradoxical function, we focused on the unique structures called HEAT repeats, which consist of repetitive amphiphilic α helices. An in vitro transport assay and FRAP analyses demonstrated that not only karyopherin β family proteins but also other proteins with HEAT repeats could pass through the NPC by themselves, and serve as transport mediators for their binding partners. Biochemical and spectroscopic analyses and molecular dynamics simulations of purified HEAT-rich proteins revealed that they interact with hydrophobic groups, including phenyl and alkyl groups, and undergo reversible conformational changes in tertiary structures, but not in secondary structures. These results show that conformational changes in the flexible amphiphilic motifs play a critical role in translocation through the NPC.

MD Simulations and FRET Reveal an Environment-Sensitive Conformational Plasticity of Importin-β

“…In fact, most crystals containing closed conformations were obtained in solutions of high PEG concentrations with a markedly decreased polarity, whereas the crystal structure obtained from a PEG-free solution showed a much more open conformation (Table S2). Similar conformational plasticity of Importin-b in response to changes in the polarity of the solvent (by addition of different types of alcohols) was observed in a recent study (57).…”

HEAT repeats – versatile arrays of amphiphilic helices working in crowded environments?

“…Karyopherins interact with the FG motifs and other hydrophobic residues of FG-Nups through a hydrophobic pocket that is formed by adjacent A-helices of their HEAT repeat (Bayliss et al, 2000(Bayliss et al, , 2002Liu and Stewart, 2005). Our recent spectroscopic analysis combined with molecular dynamics simulation of importin β has demonstrated that the structural flexibility of HEAT repeats plays a crucial role in allowing the migration through the crowded space of the nuclear pore channel and is mediated through interactions with FG motifs (Yoshimura et al, 2014). Here, a number of weak interactions between multiple FG motifs and importin β induce temporary conformational changes in both the HEAT repeat and the matrix of FG-hydrogels, which enable karyopherins to migrate through the hydrogel-like environment of the nuclear pore channel (see below for more detailed discussion).…”

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

“…Likewise, NTF2 44 as well as non-specific molecules that exert sufficient FG-repeat binding 63,64 might exhibit varying degrees of ROD-like translocation.…”