Investigation of Phosphorylation-Induced Folding of an Intrinsically Disordered Protein by Coarse-Grained Molecular Dynamics

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α-Synuclein is an intrinsically disordered protein occurring in different conformations and prone to aggregate in β-sheet structures, which are the hallmark of the Parkinson disease. Missense mutations are associated with familial forms of this neuropathy. How these single amino-acid substitutions modify the conformations of wild-type α-synuclein is unclear. Here, using coarse-grained molecular dynamics simulations, we sampled the conformational space of the wild type and mutants (A30P, A53P, and E46K) of α-synuclein monomers for an effective time scale of 29.7 ms. To characterize the structures, we developed an algorithm, CUTABI (CUrvature and Torsion based of Alpha-helix and Beta-sheet Identification), to identify residues in the α-helix and β-sheet from Cα-coordinates. CUTABI was built from the results of the analysis of 14,652 selected protein structures using the Dictionary of Secondary Structure of Proteins (DSSP) algorithm. DSSP results are reproduced with 93% of success for 10 times lower computational cost. A two-dimensional probability density map of α-synuclein as a function of the number of residues in the α-helix and β-sheet is computed for wild-type and mutated proteins from molecular dynamics trajectories. The density of conformational states reveals a two-phase characteristic with a homogeneous phase (state B, β-sheets) and a heterogeneous phase (state HB, mixture of α-helices and β-sheets). The B state represents 40% of the conformations for the wild-type, A30P, and E46K and only 25% for A53T. The density of conformational states of the B state for A53T and A30P mutants differs from the wild-type one. In addition, the mutant A53T has a larger propensity to form helices than the others. These findings indicate that the equilibrium between the different conformations of the α-synuclein monomer is modified by the missense mutations in a subtle way. The α-helix and β-sheet contents are promising order parameters for intrinsically disordered proteins, whereas other structural properties such as average gyration radius, Rg, or probability distribution of Rg cannot discriminate significantly the conformational ensembles of the wild type and mutants. When separated in states B and HB, the distributions of Rg are more significantly different, indicating that global structural parameters alone are insufficient to characterize the conformational ensembles of the α-synuclein monomer.

Section: Methodsmentioning

confidence: 99%

Missense Mutations Modify the Conformational Ensemble of the α-Synuclein Monomer Which Exhibits a Two-Phase Characteristic

Guzzo

Delarue

Rojas

et al. 2021

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“…Here, we are going a step further by using unbiased replica exchange MD simulations of two α -synuclein molecules in an implicit solvent by using a physics-based coarse-grained UNited-RESidue (UNRES) force field ( Maisuradze et al, 2010 ; Liwo et al, 2019 ) on a time scale of 29.7 milliseconds (72 replicas of 412 μ s each for each variant studied), which is three orders of magnitude larger than typical all-atom MD simulations ( Khalili et al, 2005 ). The force field was calibrated to reproduce the structure and thermodynamics of small model proteins and applied with success to simulate protein folding ( Maisuradze et al, 2010 ; Zhou et al, 2014 ; Sieradzan et al, 2021 ), large-scale conformational dynamics ( Gołaś et al, 2012 ), A β -amyloids ( Rojas et al, 2017 ), and the effect of A β -fibrils on the aggregation of tau protein ( Rojas et al, 2018 ). In the present MD simulations, most of the α -syn molecules do not aggregate and remain thus in a monomeric conformation.…”

Section: Introductionmentioning

confidence: 99%

Wild-Type α-Synuclein and Variants Occur in Different Disordered Dimers and Pre-Fibrillar Conformations in Early Stage of Aggregation

Guzzo

Delarue

Rojas

et al. 2022

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α-Synuclein is a 140 amino-acid intrinsically disordered protein mainly found in the brain. Toxic α-synuclein aggregates are the molecular hallmarks of Parkinson’s disease. In vitro studies showed that α-synuclein aggregates in oligomeric structures of several 10th of monomers and into cylindrical structures (fibrils), comprising hundred to thousands of proteins, with polymorphic cross-β-sheet conformations. Oligomeric species, formed at the early stage of aggregation remain, however, poorly understood and are hypothezised to be the most toxic aggregates. Here, we studied the formation of wild-type (WT) and mutant (A30P, A53T, and E46K) dimers of α-synuclein using coarse-grained molecular dynamics. We identified two principal segments of the sequence with a higher propensity to aggregate in the early stage of dimerization: residues 36–55 and residues 66–95. The transient α-helices (residues 53–65 and 73–82) of α-synuclein monomers are destabilized by A53T and E46K mutations, which favors the formation of fibril native contacts in the N-terminal region, whereas the helix 53–65 prevents the propagation of fibril native contacts along the sequence for the WT in the early stages of dimerization. The present results indicate that dimers do not adopt the Greek key motif of the monomer fold in fibrils but form a majority of disordered aggregates and a minority (9–15%) of pre-fibrillar dimers both with intra-molecular and intermolecular β-sheets. The percentage of residues in parallel β-sheets is by increasing order monomer < disordered dimers < pre-fibrillar dimers. Native fibril contacts between the two monomers are present in the NAC domain for WT, A30P, and A53T and in the N-domain for A53T and E46K. Structural properties of pre-fibrillar dimers agree with rupture-force atomic force microscopy and single-molecule Förster resonance energy transfer available data. This suggests that the pre-fibrillar dimers might correspond to the smallest type B toxic oligomers. The probability density of the dimer gyration radius is multi-peaks with an average radius that is 10 Å larger than the one of the monomers for all proteins. The present results indicate that even the elementary α-synuclein aggregation step, the dimerization, is a complicated phenomenon that does not only involve the NAC region.

“…Considering the recent computational successes in describing the structural features and dynamics of disordered proteins ( Cino et al, 2011 ; Krzeminski et al, 2012 ; Do et al, 2014 ; Ramis et al, 2019 ; Pietrek et al, 2020 ; Wilson et al, 2021a ; Chang et al, 2021 ), molecular simulation of specific protein segments could help to more accurately characterize Matrin3, FUS, and TDP-43, by producing a structural ensemble rather than a single frame. Computational methods have also been extended to study protein aggregation propensities ( Morriss-Andrews and Shea, 2015 ), liquid-liquid phase-separation ( Paloni et al, 2020 ; Benayad et al, 2021 ), IDP-protein interactions ( Cino et al, 2013 ; Do et al, 2016 ; Karttunen et al, 2018 ), and the effects of mutation ( Vacic et al, 2012 ; Wilson et al, 2021b ) and post-translational modification ( Jin and Graeter, 2021 ; Sieradzan et al, 2021 ) on protein structure, all potential avenues for investigation of a disordered ALS protein like Matrin3.…”

Section: Structure Disorder and Llps In Matrin3mentioning

confidence: 99%

Matrin3: Disorder and ALS Pathogenesis

Salem

Wilson

Rutledge

et al. 2022

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of both upper and lower motor neurons in the brain and spinal cord. ALS is associated with protein misfolding and inclusion formation involving RNA-binding proteins, including TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS). The 125-kDa Matrin3 is a highly conserved nuclear DNA/RNA-binding protein that is implicated in many cellular processes, including binding and stabilizing mRNA, regulating mRNA nuclear export, modulating alternative splicing, and managing chromosomal distribution. Mutations in MATR3, the gene encoding Matrin3, have been identified as causal in familial ALS (fALS). Matrin3 lacks a prion-like domain that characterizes many other ALS-associated RNA-binding proteins, including TDP-43 and FUS, however, our bioinformatics analyses and preliminary studies document that Matrin3 contains long intrinsically disordered regions that may facilitate promiscuous interactions with many proteins and may contribute to its misfolding. In addition, these disordered regions in Matrin3 undergo numerous post-translational modifications, including phosphorylation, ubiquitination and acetylation that modulate the function and misfolding of the protein. Here we discuss the disordered nature of Matrin3 and review the factors that may promote its misfolding and aggregation, two elements that might explain its role in ALS pathogenesis.