bims-indpro 2022-10-02 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–10–02
thirteen papers selected by
Sara Mingu, Johannes Gutenberg University

Anchor Residues Govern Binding and Folding of an Intrinsically Disordered Domain.
Polarizable Force Field of Intrinsically Disordered Proteins with CMAP and Reweighting Optimization.
Influence of Force Field Choice on the Conformational Landscape of Rat and Human Islet Amyloid Polypeptide.
The material properties of a bacterial-derived biomolecular condensate tune biological function in natural and synthetic systems.
The disordered MAX N-terminus modulates DNA binding of the transcription factor MYC:MAX.
Characterization of the Conformations of Amyloid Beta 42 in Solution That May Mediate Its Initial Hydrophobic Aggregation.
Confinement dependence of protein-associated solvent dynamics around different classes of proteins, from the EPR spin probe perspective.
Comparative and evolutionary analysis of Arabidopsis RIN4-like/NOI proteins induced by herbivory.
Protein conformation and biomolecular condensates.
AML-158 Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.
Self-Effected Allosteric Coupling and Cooperativity in Hypoxic Response Regulation with Disordered Proteins.
Phase separation in amino acid mixtures is governed by composition.
Protein structural transitions critically transform the network connectivity and viscoelasticity of RNA-binding protein condensates but RNA can prevent it.

ACS Chem Biol. 2022 Sep 26.

Anchor Residues Govern Binding and Folding of an Intrinsically Disordered Domain.

Haley I Merritt, Nicholas Sawyer, Andrew M Watkins, Paramjit S Arora.

Minimal protein mimics have yielded novel classes of protein-protein interaction inhibitors; however, this success has not been extended to targeting intrinsically disordered proteins, which represent a significant proportion of important therapeutic targets. We sought to determine the requirements for binding an intrinsically disordered region (IDR) by its native binding partner as a prelude to developing minimal protein mimics that regulate IDR interactions. Our analysis reinforces the hypothesis that IDRs reside on a fulcrum between unfolded and folded states and that a handful of key binding residues on partner protein surfaces dictate their folding. Our studies also suggest that minimal mimics of protein surfaces may not offer specific ligands for IDRs and that it would be more judicious to target the globular protein partners of IDRs.

DOI: https://doi.org/10.1021/acschembio.2c00619
J Chem Inf Model. 2022 Sep 30.

Polarizable Force Field of Intrinsically Disordered Proteins with CMAP and Reweighting Optimization.

Xiaochen Cui, Hao Liu, Hai-Feng Chen.

Intrinsically disordered proteins (IDPs) are highly structurally heterogeneous without a specific tertiary structure under physiology conditions and play key roles in the development of human diseases. Due to the characteristics of diverse conformations, as one of the important methods, molecular dynamics simulation can complement information for experimental methods. Because of the enrichment for charged amino acids for IDPs, polarizable force fields should be a good choice for the simulation of IDPs. However, current polarizable force fields are limited in sampling conformer features of IDPs. Therefore, a polarizable force field was released and named Drude2019IDP based on Drude2019 with reweighting and grid-based potential energy correction map optimization. In order to evaluate the performance of Drude2019IDP, 16 dipeptides, 18 short peptides, 3 representative IDPs, and 5 structural proteins were simulated. The results show that the NMR observables driven by Drude2019IDP are in better agreement with the experiment data than those by Drude2019 on short peptides and IDPs. Drude2019IDP can sample more diverse conformations than Drude2019. Furthermore, the performances of the two force fields are similar to the sample ordered proteins. These results confirm that the developed Drude2019IDP can improve the reproduction of conformers for intrinsically disordered proteins and can be used to gain insight into the paradigm of sequence-disorder for IDPs.

DOI: https://doi.org/10.1021/acs.jcim.2c00835
Proteins. 2022 Sep 26.

Influence of Force Field Choice on the Conformational Landscape of Rat and Human Islet Amyloid Polypeptide.

Sandra J Moore, Evelyne Deplazes, Ricardo L Mancera.

  Human islet amyloid polypeptide (hIAPP) is a naturally occurring, intrinsically disordered protein (IDP) whose abnormal aggregation into toxic soluble oligomers and insoluble amyloid fibrils is a pathological feature in type-2 diabetes. Rat IAPP (rIAPP) differs from hIAPP by only six amino acids yet has a reduced tendency to aggregate or form fibrils. The structures of the monomeric forms of IAPP are difficult to characterise due to their intrinsically disordered nature. Molecular dynamics simulations can provide a detailed characterisation of the monomeric forms of rIAPP and hIAPP in near-physiological conditions. In this work, the conformational landscapes of rIAPP and hIAPP as a function of secondary structure content were predicted using well-tempered bias exchange metadynamics simulations. Several combinations of commonly used biomolecular force fields and water models were tested. The predicted conformational preferences of both rIAPP and hIAPP are typical of IDPs, exhibiting dominant random coil structures but showing a low propensity for transient α-helical conformations. Predicted NMR Cα chemical shifts reveal different preferences with each force field towards certain conformations, with AMBERff99SBnmr2/TIP4Pd showing the best agreement with the experiment. Comparisons of secondary structure content demonstrate residue-specific differences between hIAPP and rIAPP that may reflect their different aggregation propensities. This article is protected by copyright. All rights reserved.

Keywords:  intrinsically disordered proteins; islet amyloid polypeptide (IAPP); metadynamics; secondary structure

DOI:  https://doi.org/10.1002/prot.26432
Nat Commun. 2022 Sep 26. 13(1): 5643

The material properties of a bacterial-derived biomolecular condensate tune biological function in natural and synthetic systems.

Keren Lasker, Steven Boeynaems, Vinson Lam, Daniel Scholl, Emma Stainton, Adam Briner, Maarten Jacquemyn, Dirk Daelemans, Ashok Deniz, Elizabeth Villa, Alex S Holehouse, Aaron D Gitler, Lucy Shapiro.

Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells.

DOI: https://doi.org/10.1038/s41467-022-33221-z
J Mol Biol. 2022 Sep 26. pii: S0022-2836(22)00453-3. [Epub ahead of print] 167833

The disordered MAX N-terminus modulates DNA binding of the transcription factor MYC:MAX.

Stefan Schütz, Christian Bergsdorf, Benedikt Goretzki, Andreas Lingel, Martin Renatus, Alvar D Gossert, Wolfgang Jahnke.

  The intrinsically disordered protein MYC belongs to the family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors (TFs). In complex with its cognate binding partner MAX, MYC preferentially binds to E-Box promotor sequences where it controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. Intramolecular regulation of MYC:MAX has not yet been investigated in detail. In this work, we use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map interactions between the disordered MAX N-terminus and the MYC:MAX DNA binding domain (DBD). We find that this binding event is mainly driven by electrostatic interactions and that it is competitive with DNA binding. Using Nuclear Magnetic resonance (NMR) spectroscopy and Surface Plasmon Resonance (SPR), we demonstrate that the MAX N-terminus serves to accelerate DNA binding kinetics of MYC:MAX and MAX:MAX dimers, while it simultaneously provides specificity for E-Box DNA. We also establish that these effects are further enhanced by Casein Kinase 2-mediated phosphorylation of two serine residues in the MAX N-terminus. Our work provides new insights how bHLH-LZ TFs are regulated by intramolecular interactions between disordered regions and the folded DNA binding domain.

Keywords:  E-Box; bHLH-LZ; intramolecular interaction; molecular mechanism; phosphorylation

DOI:  https://doi.org/10.1016/j.jmb.2022.167833
J Phys Chem B. 2022 Sep 30.

Characterization of the Conformations of Amyloid Beta 42 in Solution That May Mediate Its Initial Hydrophobic Aggregation.

Krushna Sonar, Ricardo L Mancera.

Intrinsically disordered peptides, such as amyloid β42 (Aβ42), lack a well-defined structure in solution. Aβ42 can undergo abnormal aggregation and amyloidogenesis in the brain, forming fibrillar plaques, a hallmark of Alzheimer's disease. The insoluble fibrillar forms of Aβ42 exhibit well-defined, cross β-sheet structures at the molecular level and are less toxic than the soluble, intermediate disordered oligomeric forms. However, the mechanism of initial interaction of monomers and subsequent oligomerization is not well understood. The structural disorder of Aβ42 adds to the challenges of determining the structural properties of its monomers, making it difficult to understand the underlying molecular mechanism of pathogenic aggregation. Certain regions of Aβ42 are known to exhibit helical propensity in different physiological conditions. NMR spectroscopy has shown that the Aβ42 monomer at lower pH can adopt an α-helical conformation and as the pH is increased, the peptide switches to β-sheet conformation and aggregation occurs. CD spectroscopy studies of aggregation have shown the presence of an initial spike in the amount of α-helical content at the start of aggregation. Such an increase in α-helical content suggests a mechanism wherein the peptide can expose critical non-polar residues for interaction, leading to hydrophobic aggregation with other interacting peptides. We have used molecular dynamics simulations to characterize in detail the conformational landscape of monomeric Aβ42 in solution to identify molecular properties that may mediate the early stages of oligomerization. We hypothesized that conformations with α-helical structure have a higher probability of initiating aggregation because they increase the hydrophobicity of the peptide. Although random coil conformations were found to be the most dominant, as expected, α-helical conformations are thermodynamically accessible, more so than β-sheet conformations. Importantly, for the first time α-helical conformations are observed to increase the exposure of aromatic and hydrophobic residues to the aqueous solvent, favoring their hydrophobically driven interaction with other monomers to initiate aggregation. These findings constitute a first step toward characterizing the mechanism of formation of disordered, low-order oligomers of Aβ42.

DOI: https://doi.org/10.1021/acs.jpcb.2c04743
Phys Chem Chem Phys. 2022 Sep 27.

Confinement dependence of protein-associated solvent dynamics around different classes of proteins, from the EPR spin probe perspective.

Wei Li, Katie Lynn Whitcomb, Kurt Warncke.

Protein function is modulated by coupled solvent fluctuations, subject to the degree of confinement from the surroundings. To identify universal features of the external confinement effect, the temperature dependence of the dynamics of protein-associated solvent over 200-265 K for proteins representative of different classes and sizes is characterized by using the rotational correlation time (detection bandwidth, 10-10-10-7 s) of the electron paramagnetic resonance (EPR, X-band) spin probe, TEMPOL, which is restricted to regions vicinal to protein in frozen aqueous solution. Weak (protein surrounded by aqueous-dimethylsulfoxide cryosolvent mesodomain) and strong (no added crysolvent) conditions of ice boundary confinement are imposed. The panel of soluble proteins represents large and small oligomeric (ethanolamine ammonia-lyase, 488 kDa; streptavidin, 52.8 kDa) and monomeric (myoglobin, 16.7 kDa) globular proteins, an intrinsically disordered protein (IDP, β-casein, 24.0 kDa), an unstructured peptide (protamine, 4.38 kDa) and a small peptide with partial backbone order (amyloid-β residues 1-16, 1.96 kDa). Expanded and condensate structures of β-casein and protamine are resolved by the spin probe under weak and strong confinement, respectively. At each confinement condition, the soluble globular proteins display common T-dependences of rotational correlation times and normalized weights, for two mobility components, protein-associated domain, PAD, and surrounding mesodomain. Strong confinement induces a detectable PAD component and emulation of globular protein T-dependence by the amyloid-β peptide. Confinement uniformly impacts soluble globular protein PAD dynamics, and is therefore a generic control parameter for modulation of soluble globular protein function.

DOI: https://doi.org/10.1039/d2cp03047k
PLoS One. 2022 ;17(9): e0270791

Comparative and evolutionary analysis of Arabidopsis RIN4-like/NOI proteins induced by herbivory.

Estefania Contreras, Manuel Martinez.

The spider mite Tetranychus urticae is an economically important agricultural pest, which feeds on a broad spectrum of plant species. In an RNAseq experiment performed in our laboratory, 4 of the 15 members of the RIN4-like/NOI family of Arabidopsis thaliana were significantly overexpressed after T. urticae infestation. Two of them (NOI3 and NOI5) are shorter and harbour one NOI domain, which characterises this family, and the other two (NOI10 and NOI11) have two-NOI domains. The only member of this family characterized is RIN4, a two-NOI intrinsically disordered protein anchored to the plasma membrane and involved in plant defence against bacterial pathogens. The function of all other members of the RIN4-like/NOI Arabidopsis family and their putative role in herbivore defence remains unknown. We perform a comparative genomic analysis of RIN4-like/NOI sequences to study the evolutionary features of this protein family and the distribution of its members among species. We show that short one-NOI proteins were more numerous and exhibited lower disorder propensity compared to two-NOI members. NOI10 and NOI11, from the two-NOI group, are included in a clade-specific expansion of Brassicaceae with unique predicted posttranslational modification sites and clear predicted structural differences from RIN4. Our analysis suggests that the members of the RIN4-like/NOI family upregulated after mite feeding have novel functions different from those assigned to RIN4, likely involving adaptation to stress specialisation.

DOI: https://doi.org/10.1371/journal.pone.0270791
Curr Res Struct Biol. 2022 ;4 285-307

Protein conformation and biomolecular condensates.

Diego S Vazquez, Pamela L Toledo, Alejo R Gianotti, Mario R Ermácora.

  Protein conformation and cell compartmentalization are fundamental concepts and subjects of vast scientific endeavors. In the last two decades, we have witnessed exciting advances that unveiled the conjunction of these concepts. An avalanche of studies highlighted the central role of biomolecular condensates in membraneless subcellular compartmentalization that permits the spatiotemporal organization and regulation of myriads of simultaneous biochemical reactions and macromolecular interactions. These studies have also shown that biomolecular condensation, driven by multivalent intermolecular interactions, is mediated by order-disorder transitions of protein conformation and by protein domain architecture. Conceptually, protein condensation is a distinct level in protein conformational landscape in which collective folding of large collections of molecules takes place. Biomolecular condensates arise by the physical process of phase separation and comprise a variety of bodies ranging from membraneless organelles to liquid condensates to solid-like conglomerates, spanning lengths from mesoscopic clusters (nanometers) to micrometer-sized objects. In this review, we summarize and discuss recent work on the assembly, composition, conformation, material properties, thermodynamics, regulation, and functions of these bodies. We also review the conceptual framework for future studies on the conformational dynamics of condensed proteins in the regulation of cellular processes.

Keywords:  Intrinsically disordered proteins; Membraneless organelles; Mesoscopic clusters; Nanocondensates; Phase separation; Protein coacervates; Protein colloids; Protein condensates; Protein conformation; Protein folding

DOI:  https://doi.org/10.1016/j.crstbi.2022.09.004
Clin Lymphoma Myeloma Leuk. 2022 Oct;pii: S2152-2650(22)01234-4. [Epub ahead of print]22 Suppl 2 S220

AML-158 Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

Nicole Michmerhuizen, Bappaditya Chandra, Hazheen Shirnekhi, Swarnendu Tripathi, Brittany Pioso, David Baggett, Diana Mitrea, Ilaria Lacobucci, Michael White, Jingjing Chen, Cheon-Gil Park, Huiyun Wu, Stanley Pounds, Anna Medyukhina, Khaled Khairy, Qingsong Gao, Chunxu Qu, Scott Gorman, Simran Bawa, Carolyn Maslanka, Swati Kinger, Priyanka Dogra, Danika Di Giacomo, Cristina Mecucci, Jeffery Klco, Charles Mullighan, Richard Kriwacki.

   CONTEXT: Rearrangements of the nucleoporin 98 gene (NUP98) define a high-risk subset of childhood acute myeloid leukemia (AML). The resulting fusion oncoproteins (FOs) involve the N-terminal, intrinsically disordered region of NUP98, and the C-terminal portion of one of more than 30 identified fusion partners. Approximately one third of fusion partners have DNA-binding homeodomains, and the remaining partners have other domains involved in gene regulation.
OBJECTIVE: NUP98 FOs have long been known to localize in nuclear puncta. Here, we investigated whether these puncta form by liquid-liquid phase separation (LLPS) and how they might contribute to cell transformation.
DESIGN: We first focused on the NUP98::HOXA9 (NHA9) FO, in which the HOXA9 fusion partner includes a DNA-binding homeodomain. We expressed GFP-tagged NHA9 in HEK293T cells and characterized the resulting FO-associated nuclear puncta. We also investigated lentiviral FO expression in mouse hematopoietic stem and progenitor cells (HSPCs), studying localization using confocal imaging, self-renewal using colony forming unit assays, and gene expression using RNA sequencing. We next mutated the phenylalanine glycine (FG) repeats of NUP98 to disrupt interactions with other FOs and interacting proteins or mutated the homeodomain of HOXA9 to disrupt DNA binding. Finally, we investigated the applicability of our findings to other NUP98 FOs with and without homeodomains.
RESULTS: NHA9 localizes in nuclear puncta in HEK293T and HSPCs, and mutation of FG repeats or the HOXA9 homeodomain abrogates puncta formation. Furthermore, expression of NHA9 confers self-renewal, but cell transformation does not occur in FO mutants that fail to form puncta. Upregulation of NUP98 FO target genes, including the HOXA cluster, occurred with expression of NHA9 and transforming, puncta-positive mutants but not in mutant cells that did not form puncta or transform HSPCs. Expression of additional NUP98 FOs (including NUP98::KDM5A, NUP98::LNP1, and NUP98::PRRX1) led to both puncta formation and cell transformation. Finally, cells from a NUP98::KDM5A patient derived xenograft confirmed puncta formation with endogenous FO expression.
CONCLUSIONS: Taken together, these results demonstrate that NUP98 FOs undergo LLPS, show that phase separation is critical for cell transformation and gene expression changes, and provide rationale to further explore the exploitation of NUP98 FO-associated puncta for therapeutical benefit.

Keywords:  AML; NUP98; acute myeloid leukemia; fusion oncoprotein; phase separation

DOI:  https://doi.org/10.1016/S2152-2650(22)01234-4
J Phys Chem Lett. 2022 Sep 28. 9201-9209

Self-Effected Allosteric Coupling and Cooperativity in Hypoxic Response Regulation with Disordered Proteins.

Bin Wen, Weiwei Zhang, Yangyang Zhang, Hai Lei, Yi Cao, Wenfei Li, Wei Wang.

Hypersensitive regulation of cellular hypoxic response relies on cooperative displacement of one disordered protein (HIF-1α) by another disordered protein (CITED2) from the target in a negative feedback loop. Considering the weak intramolecule coupling in disordered proteins, the molecular mechanism of high cooperativity in the molecular displacement event remains elusive. Herein, we show that disordered proteins utilize a "self-effected allostery" mechanism to achieve high binding cooperativity. Different from the conventional allostery mechanisms shown by many structured or disordered proteins, this mechanism utilizes one part of the disordered protein as the effector to trigger the allosteric coupling and enhance the binding of the remaining part of the same disordered protein, contributing to high cooperativity of the displacement event. The conserved charge motif of CITED2 is the key determinant of the molecular displacement event by serving as the effector of allosteric coupling. Such self-effected allostery provides an efficient strategy to achieve high cooperativity in the molecular events involving disordered proteins.

DOI: https://doi.org/10.1021/acs.jpclett.2c02065
Biophys J. 2022 Sep 29. pii: S0006-3495(22)00781-0. [Epub ahead of print]

Phase separation in amino acid mixtures is governed by composition.

David De Sancho.

Macromolecular phase separation has recently come to immense prominence as it is central to the formation of membraneless organelles, leading to a new paradigm of cellular organization. This type of phase transition, often termed liquid-liquid phase separation (LLPS), is mediated by molecular interactions between biomolecules, including nucleic acids and both ordered and disordered proteins. In the latter case, the separation between protein-dense and dilute phases is often interpreted using models adapted from polymer theory. Specifically, the "stickers and spacers" model proposes that the formation of condensate-spanning networks in protein solutions originates from the interplay between two classes of residues and that the main determinants for phase separation are multivalency and sequence patterning. The duality of roles of stickers (aromatics like Phe and Tyr) and spacers (Gly and polar residues) may apply more broadly in protein-like mixtures, and the presence of these two types of components alone may suffice for LLPS to take place. In order to explore this hypothesis, we use atomistic molecular dynamics simulations of capped amino-acid residues as a minimal model system. We study the behaviour of pure amino acids in water for three types of residues corresponding to the spacer and sticker categories, and their multicomponent mixtures. In agreement with previous observations, we find that the spacer-type amino acids fail to phase-separate on their own, while the sticker is prone to aggregation. However, ternary amino acid mixtures involving both types of amino acids phase-separate into two phases that retain intermediate degrees of compaction and greater fluidity than sticker-only condensates. Our results suggest that LLPS is an emergent property of amino acid mixtures determined by composition.

DOI: https://doi.org/10.1016/j.bpj.2022.09.031
Nat Commun. 2022 Sep 29. 13(1): 5717

Protein structural transitions critically transform the network connectivity and viscoelasticity of RNA-binding protein condensates but RNA can prevent it.

Andres R Tejedor, Ignacio Sanchez-Burgos, Maria Estevez-Espinosa, Adiran Garaizar, Rosana Collepardo-Guevara, Jorge Ramirez, Jorge R Espinosa.

Biomolecular condensates, some of which are liquid-like during health, can age over time becoming gel-like pathological systems. One potential source of loss of liquid-like properties during ageing of RNA-binding protein condensates is the progressive formation of inter-protein β-sheets. To bridge microscopic understanding between accumulation of inter-protein β-sheets over time and the modulation of FUS and hnRNPA1 condensate viscoelasticity, we develop a multiscale simulation approach. Our method integrates atomistic simulations with sequence-dependent coarse-grained modelling of condensates that exhibit accumulation of inter-protein β-sheets over time. We reveal that inter-protein β-sheets notably increase condensate viscosity but does not transform the phase diagrams. Strikingly, the network of molecular connections within condensates is drastically altered, culminating in gelation when the network of strong β-sheets fully percolates. However, high concentrations of RNA decelerate the emergence of inter-protein β-sheets. Our study uncovers molecular and kinetic factors explaining how the accumulation of inter-protein β-sheets can trigger liquid-to-solid transitions in condensates, and suggests a potential mechanism to slow such transitions down.

DOI: https://doi.org/10.1038/s41467-022-32874-0