Determining Binding Kinetics of Intrinsically Disordered Proteins by NMR Spectroscopy

In the nuclear pore complex (NPC), intrinsically disordered proteins (FG Nups) along with their interactions with more globular proteins called nuclear transport receptors (NTRs) are vital to the selectivity of transport into and out of the cell nucleus. While such interactions can be modelled at different levels of coarse graining, in-vitro experimental data have been quantitatively described by minimal models that describe FG Nups as cohesive homogeneous polymers and NTRs as uniformly cohesive spheres, where the heterogeneous effects have been smeared out. By definition, these minimal models do not account for the explicit heterogeneities in FG Nup sequences, essentially a string of cohesive and non-cohesive polymer units, and at the NTR surface. Here, we develop computational and analytical models that do take into account such heterogeneity at a level of minimal complexity, and compare them to experimental data on single-molecule interactions between FG Nups and NTRs. Overall, we find that the heterogeneous nature of FG Nups and NTRs plays a minor role for their equilibrium binding properties, but is of significance when it comes to (un)binding kinetics. Using our models, we predict how binding equilibria and kinetics depend on the distribution of cohesive blocks in the FG Nup sequences and of the binding pockets at the NTR surface, with multivalency playing a key role. Finally, we observe that single-molecule binding kinetics has a rather minor influence on the diffusion of NTRs in polymer melts consisting of FG-Nup-like sequences.

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Towards Targeting the Disordered SARS-CoV-2 Nsp2 C-terminal Region: Partial Structure and Dampened Mobility Revealed by NMR Spectroscopy

10.1101/2020.11.09.374173 ◽

2020 ◽

Author(s):

Miguel Mompeán ◽

Miguel Á. Treviño ◽

Douglas V. Laurents

Keyword(s):

Nmr Spectroscopy ◽

Drug Targets ◽

Intrinsically Disordered Proteins ◽

Small Population ◽

Terminal Region ◽

Disordered Proteins ◽

X Ray Crystallography ◽

Intrinsically Disordered ◽

Level Information ◽

Human Proteins

AbstractIntrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many virus use their own IDPs to “hack” these processes to disactive host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational perferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2 interacts with human proteins that regulate translation initiation and endosome vesicle sorting, and the C-terminal region of this protein is predicted to be disordered. Molecules that block these interactions could be valuable leads for drug development. To enable inhibitor screening and to uncover conformational preferences and dynamics, we have expressed and purified the 13C,15N-labeled C-terminal region of Nsp2. The 13Cβ and backbone 13CO, 1HN, 13Cα and 15N nuclei were assigned by analysis of a series of 2D 1H-15N HSQC and 13C-15N CON as well as 3D HNCO, HNCA, CBCAcoNH and HncocaNH spectra. Overall, the chemical shift data confirm that this region is chiefly disordered, but contains two five-residue segments that adopt a small population of β-strand structure. Whereas the region is flexible on ms/ms timescales as gauged by T1ρ measurements, the {1H}-15N NOEs reveal a flexibility on ns/ps timescales that is midway between a fully flexible and a completely rigid chain.

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H-start for exclusively heteronuclear NMR spectroscopy: The case of intrinsically disordered proteins

Journal of Magnetic Resonance ◽

10.1016/j.jmr.2009.02.012 ◽

2009 ◽

Vol 198 (2) ◽

pp. 275-281 ◽

Cited By ~ 62

Author(s):

Wolfgang Bermel ◽

Ivano Bertini ◽

Veronika Csizmok ◽

Isabella C. Felli ◽

Roberta Pierattelli ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Intrinsically Disordered Proteins ◽

Heteronuclear Nmr ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Heteronuclear Nmr Spectroscopy

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