The development of AlphaFold2 marked a paradigm-shift in the structural biology community. Herein, we assess the ability of AlphaFold2 to predict disordered regions against traditional sequence-based disorder predictors. We find that AlphaFold2 performs well at discriminating disordered regions, but also note that the disorder predictor one constructs from an AlphaFold2 structure determines accuracy. In particular, a naïve, but non-trivial assumption that residues assigned to helices, strands, and H-bond stabilized turns are likely ordered and all other residues are disordered results in a dramatic overestimation in disorder; conversely, the predicted local distance difference test (pLDDT) provides an excellent measure of residue-wise disorder. Furthermore, by employing molecular dynamics (MD) simulations, we note an interesting relationship between the pLDDT and secondary structure, that may explain our observations and suggests a broader application of the pLDDT for characterizing the local dynamics of intrinsically disordered proteins and regions (IDPs/IDRs).
The development of AlphaFold2 was a paradigm-shift in the structural biology community; herein we assess the ability of AlphaFold2 to predict disordered regions against traditional sequence-based disorder predictors. We find that a naive use of Dictionary of Secondary Structure of Proteins (DSSP) to separate ordered from disordered regions leads to a dramatic overestimation in disorder content, and that the Predicted Aligned Error (PAE) provides a much more rigorous metric. In addition, we show that even when used for disorder prediction, conventional predictors can outperform the PAE in disorder identification, and note an interesting relationship between the PAE and secondary structure that may explain our observations and hints at a broader application of the PAE to IDP dynamics.
The nuclear factor erythroid 2-related factor 2 (Nrf2)-ARE transcriptional response pathway plays a critical role in protecting the cell from oxidative stresses via the upregulation of cytoprotective genes. Aberrant activation of Nrf2 in cancer cells can confer this cytoprotectivity, thereby reducing the efficacy of both chemotherapeutics and radiotherapies. Key to this antioxidant pathway is the interaction between Nrf2 and CREB binding protein (CBP), mediated by the Neh4 and Neh5 domains of Nrf2. Disruption of this interaction via small-molecule therapeutics could negate the effects of aberrant Nrf2 upregulation. Due to the disordered nature of these domains, there remains no threedimensional structure of Neh4 or Neh5, making structure-based drug design a challenge. Here, we performed 48 μs of unbiased molecular dynamics (MD) simulations with the Amber99SB*-ILDNP and CHARMM36m force fields and circular dichroism (CD) spectropolarimetry experiments to elucidate the free-state structures of these domains; no previous data regarding their conformational landscapes exists. There are two main findings: First, we find Neh5 to be markedly more disordered than Neh4, which has nine residues in the middle of the domain showing α-helical propensity, thus pointing to Neh4 and Neh5 having different binding mechanisms. Second, the two force fields show strong differences for the glutamic acid-rich Neh5 peptide but are in reasonable agreement for Neh4, which has no glutamic acid. The CHARMM36m force field agrees more closely with the CD results.
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.
We have performed 280 μs of unbiased molecular dynamics (MD) simulations to investigate the effects of 12 different cancer mutations on Kelch-like ECH-associated protein 1 (KEAP1) (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C, R470C, R470H, R470S and G476R), one of the frequently mutated proteins in lung cancer. The aim was to provide structural insight into the effects of these mutants, including a new class of ANCHOR (additionally NRF2-complexed hypomorph) mutant variants. Our work provides additional insight into the structural dynamics of mutants that could not be analyzed experimentally, painting a more complete picture of their mutagenic effects. Notably, blade-wise analysis of the Kelch domain points to stability as a possible target of cancer in KEAP1. Interestingly, structural analysis of the R470C ANCHOR mutant, the most prevalent missense mutation in KEAP1, revealed no significant change in structural stability or NRF2 binding site dynamics, possibly indicating an covalent modification as this mutant’s mode of action.
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