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

Abstract

The nuclear pore complex mediates nucleocytoplasmic transport of macromolecules in eukaryotic cells. Transport through the pore is restricted by a hydrophobic selectivity filter comprising disordered phenylalanine-glycine-rich repeats of nuclear pore proteins. Exchange through the pore requires specialized transport receptors, called exportins and importins, that interact with cargo proteins in a RanGTP-dependent manner. These receptors are highly flexible superhelical structures composed of HEAT-repeat motifs that adopt various degrees of extension in crystal structures. Here, we performed molecular-dynamics simulations using crystal structures of Importin-β in its free form or in complex with nuclear localization signal peptides as the starting conformation. Our simulations predicted that initially compact structures would adopt extended conformations in hydrophilic buffers, while contracted conformations would dominate in more hydrophobic solutions, mimicking the environment of the nuclear pore. We confirmed this experimentally by Förster resonance energy transfer experiments using dual-fluorophore-labeled Importin-β. These observations explain seemingly contradictory crystal structures and suggest a possible mechanism for cargo protection during passage of the nuclear pore. Such hydrophobic switching may be a general principle for environmental control of protein function.

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).…”

The use of unnatural amino acids to study and engineer protein function

“…Further optimization of the procedures will be necessary to extend this approach to more challenging targets. The combination of labelling via UAAs with conventional cysteine chemistry, native chemical ligation or fluorescent tags is technically less demanding and has been used to study, for example, the dynamics of nucleoporins (Figure 4a) [64 ] and nuclear transport receptors [65].…”

Impact of the crystallization condition on importin-β conformation

“…These binding areas might extend in the presence of PEG, which releases strain from the hydrophobic binding pockets of Imp and thereby affects its extension, which results in the opposite effect for Imp during SAXS, namely its spatial expansion in the hydrophobic environment. Recently, a study was published which also deals with the adaptation of Imp to its environment but resulted in the opposite findings (Halder et al, 2015). Halder and coworkers examined the conformational changes of Imp with the environment via MD simulations and FRET.…”