Impact of the crystallization condition on importin-β conformation

Abstract

In eukaryotic cells, the exchange of macromolecules between the nucleus and cytoplasm is highly selective and requires specialized soluble transport factors. Many of them belong to the importin-β superfamily, the members of which share an overall superhelical structure owing to the tandem arrangement of a specific motif, the HEAT repeat. This structural organization leads to great intrinsic flexibility, which in turn is a prerequisite for the interaction with a variety of proteins and for its transport function. During the passage from the aqueous cytosol into the nucleus, the receptor passes the gated channel of the nuclear pore complex filled with a protein meshwork of unknown organization, which seems to be highly selective owing to the presence of FG-repeats, which are peptides with hydrophobic patches. Here, the structural changes of free importin-β from a single organism, crystallized in polar (salt) or apolar (PEG) buffer conditions, are reported. This allowed analysis of the structural changes, which are attributable to the surrounding milieu and are not affected by bound interaction partners. The importin-β structures obtained exhibit significant conformational changes and suggest an influence of the polarity of the environment, resulting in an extended conformation in the PEG condition. The significance of this observation is supported by SAXS experiments and the analysis of other crystal structures of importin-β deposited in the Protein Data Bank.

Curvature of designed armadillo repeat proteins allows modular peptide binding

“…In this study, we show that dArmRPs adopt different curvature parameters in different crystal structures, sometimes even within the same AU. This demonstrates an intrinsic flexibility of the scaffold which was also observed for nArmRP (Huber et al, 1997) and importin-β, belonging to the closely related family of HEAT repeat proteins (Halder et al, 2015;Tauchert et al, 2016;Zachariae and Grubmüller, 2008). However, we expect the flexibility to be smaller in dArmRPs since the scaffold is more regular and stable compared to nArmRP (Madhurantakam et al, 2012;Parmeggiani et al, 2008;Reichen et al, 2016b).…”

Karyopherins regulate nuclear pore complex barrier and transport function

“…S4 C) with the exception of cNup153, which binds Kapα weakly, i.e., K d = 1.3 ± 0.1 µM because of an NLS-like sequence at its C terminus ( Makise et al, 2012 ; Ogawa et al, 2012 ). Nevertheless, Kapα·Kapβ1 complexes seem to be less flexible than free Kapβ1 molecules, and this might serve to stabilize the binding of Kapα·Kapβ1 to the FG Nups ( Cingolani et al, 2000 ; Tauchert et al, 2016 ). Still, MG-NLS·Kapα·Kapβ1–FG Nup binding is not measurably stronger than Kapα·Kapβ1 ( Fig.…”

Investigating Cellular Nanoscale with X-rays

“…D.1e) curves match very well as well as their pddf, which indicates different conformations of the protein in the different buffers. These results have been published in Acta Crystallographica Section D [245].…”