Thermodynamic stability of hnRNP A1 low complexity domain revealed by high‐pressure NMR

We have investigated the pressure‐ and temperature‐induced conformational changes associated with the low complexity domain of hnRNP A1, an RNA‐binding protein able to phase separate in response to cellular stress. Solution NMR spectra of the hnRNP A1 low‐complexity domain fused with protein‐G B1 domain were collected from 1 to 2500 bar and from 268 to 290 K. While the GB1 domain shows the typical pressure‐induced and cold temperature‐induced unfolding expected for small globular domains, the low‐complexity domain of hnRNP A1 exhibits unusual pressure and temperature dependences. We observed that the low‐complexity domain is pressure sensitive, undergoing a major conformational transition within the prescribed pressure range. Remarkably, this transition has the inverse temperature dependence of a typical folding‐unfolding transition. Our results suggest the presence of a low‐lying extended and fully solvated state(s) of the low‐complexity domain that may play a role in phase separation. This study highlights the exquisite sensitivity of solution NMR spectroscopy to observe subtle conformational changes and illustrates how pressure perturbation can be used to determine the properties of metastable conformational ensembles.

[1]  Jeffrey D Levengood,et al.  Author response for "Thermodynamic stability of hnRNP A1 low complexity domain revealed by high‐pressure NMR" , 2020 .

[2]  M. P. Hughes,et al.  CryoEM structure of the low-complexity domain of hnRNPA2 and its conversion to pathogenic amyloid , 2020, Nature Communications.

[3]  R. Pappu,et al.  Valence and patterning of aromatic residues determine the phase behavior of prion-like domains , 2020, Science.

[4]  A. Bax,et al.  Observation of β-Amyloid Peptide Oligomerization by Pressure-Jump NMR Spectroscopy. , 2019, Journal of the American Chemical Society.

[5]  Nicolas L. Fawzi,et al.  TDP-43 α-helical structure tunes liquid–liquid phase separation and function , 2019, Proceedings of the National Academy of Sciences.

[6]  Xueming Li,et al.  Structural basis for reversible amyloids of hnRNPA1 elucidates their role in stress granule assembly , 2019, Nature Communications.

[7]  H. Chan,et al.  Pressure-Sensitive and Osmolyte-Modulated Liquid-Liquid Phase Separation of Eye-Lens γ-Crystallins. , 2019, Journal of the American Chemical Society.

[8]  Mahdi Muhammad Moosa,et al.  Interplay Between Short-range Attraction and Long-range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein-RNA Complexes , 2019, bioRxiv.

[9]  Jeffrey D Levengood,et al.  Idiosyncrasies of hnRNP A1-RNA recognition: Can binding mode influence function. , 2019, Seminars in cell & developmental biology.

[10]  C. Royer,et al.  Lessons from pressure denaturation of proteins , 2018, Journal of The Royal Society Interface.

[11]  U. Weininger,et al.  Equilibrium and Kinetic Unfolding of GB1: Stabilization of the Native State by Pressure. , 2018, The journal of physical chemistry. B.

[12]  H. Chan,et al.  Pressure-Induced Dissolution and Reentrant Formation of Condensed, Liquid-Liquid Phase-Separated Elastomeric α-Elastin. , 2018, Chemistry.

[13]  D. Svergun,et al.  Consensus Bayesian assessment of protein molecular mass from solution X-ray scattering data , 2018, Scientific Reports.

[14]  C. Holt,et al.  FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions , 2018, Cell.

[15]  A. Bax,et al.  Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell , 2018, Proceedings of the National Academy of Sciences.

[16]  T. Mittag,et al.  Relationship of Sequence and Phase Separation in Protein Low-Complexity Regions. , 2018, Biochemistry.

[17]  Hue Sun Chan,et al.  Theories for Sequence-Dependent Phase Behaviors of Biomolecular Condensates. , 2018, Biochemistry.

[18]  R. Vernon,et al.  Pi-Pi contacts are an overlooked protein feature relevant to phase separation , 2018, eLife.

[19]  G. Makhatadze,et al.  Molecular Determinants of Temperature Dependence of Protein Volume Change upon Unfolding. , 2017, The journal of physical chemistry. B.

[20]  P. V. Konarev,et al.  ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions , 2017, Journal of applied crystallography.

[21]  L. M. Nguyen,et al.  High-pressure NMR techniques for the study of protein dynamics, folding and aggregation. , 2017, Journal of magnetic resonance.

[22]  G. Makhatadze,et al.  Molecular determinant of the effects of hydrostatic pressure on protein folding stability , 2017, Nature Communications.

[23]  S. M. Lewis,et al.  Methylarginines within the RGG-Motif Region of hnRNP A1 Affect Its IRES Trans-Acting Factor Activity and Are Required for hnRNP A1 Stress Granule Localization and Formation. , 2017, Journal of molecular biology.

[24]  Nicolas L. Fawzi,et al.  ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain. , 2016, Structure.

[25]  R. Parker,et al.  Principles and Properties of Stress Granules. , 2016, Trends in cell biology.

[26]  David R. Liu,et al.  Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein. , 2016, Molecular cell.

[27]  Roy Parker,et al.  Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins. , 2015, Molecular cell.

[28]  Jeffrey D Levengood,et al.  The First Crystal Structure of the UP1 Domain of hnRNP A1 Bound to RNA Reveals a New Look for an Old RNA Binding Protein. , 2015, Journal of molecular biology.

[29]  A. Kanagaraj,et al.  Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization , 2015, Cell.

[30]  G. Clore,et al.  The energetics of a three-state protein folding system probed by high-pressure relaxation dispersion NMR spectroscopy. , 2015, Angewandte Chemie.

[31]  Jerson L. Silva,et al.  A hypothesis to reconcile the physical and chemical unfolding of proteins , 2015, Proceedings of the National Academy of Sciences.

[32]  R. Best,et al.  Role of solvation in pressure-induced helix stabilization. , 2014, The Journal of chemical physics.

[33]  Fumio Hirata,et al.  Cavity as a source of conformational fluctuation and high-energy state: high-pressure NMR study of a cavity-enlarged mutant of T4 lysozyme. , 2014, Biophysical journal.

[34]  A Joshua Wand,et al.  Role of cavities and hydration in the pressure unfolding of T4 lysozyme , 2014, Proceedings of the National Academy of Sciences.

[35]  T. Kiefhaber,et al.  Transition state and ground state properties of the helix–coil transition in peptides deduced from high-pressure studies , 2013, Proceedings of the National Academy of Sciences.

[36]  Ad Bax,et al.  Impact of Hydrostatic Pressure on an Intrinsically Disordered Protein: A High‐Pressure NMR Study of α‐Synuclein , 2013, Chembiochem : a European journal of chemical biology.

[37]  A. Garcia,et al.  Effect of internal cavities on folding rates and routes revealed by real-time pressure-jump NMR spectroscopy. , 2013, Journal of the American Chemical Society.

[38]  M. Caputi,et al.  hnRNP A1: The Swiss Army Knife of Gene Expression , 2013, International journal of molecular sciences.

[39]  H. Soreq,et al.  Heterogeneous nuclear ribonucleoprotein A1 in health and neurodegenerative disease: From structural insights to post-transcriptional regulatory roles , 2013, Molecular and Cellular Neuroscience.

[40]  A. Garcia,et al.  Remodeling of the folding free energy landscape of staphylococcal nuclease by cavity-creating mutations. , 2012, Biochemistry.

[41]  S. Grzesiek,et al.  Key stabilizing elements of protein structure identified through pressure and temperature perturbation of its hydrogen bond network. , 2012, Nature chemistry.

[42]  Jimin Pei,et al.  Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels , 2012, Cell.

[43]  Jose A. Caro,et al.  Cavities determine the pressure unfolding of proteins , 2012, Proceedings of the National Academy of Sciences.

[44]  J. Tainer,et al.  Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. , 2011, Biopolymers.

[45]  D. Barrick,et al.  Size and sequence and the volume change of protein folding. , 2011, Journal of the American Chemical Society.

[46]  Greg L. Hura,et al.  X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. , 2011, Quarterly reviews of biophysics.

[47]  R. Winter,et al.  High-pressure SAXS study of folded and unfolded ensembles of proteins. , 2010, Biophysical journal.

[48]  Pau Bernadó,et al.  A self-consistent description of the conformational behavior of chemically denatured proteins from NMR and small angle scattering. , 2009, Biophysical journal.

[49]  C. Royer,et al.  Towards a quantitative understanding of protein hydration and volumetric properties. , 2008, Chemphyschem : a European journal of chemical physics and physical chemistry.

[50]  David J Wilton,et al.  Pressure‐induced changes in the solution structure of the GB1 domain of protein G , 2007, Proteins.

[51]  L. Kay,et al.  Probing the transition state ensemble of a protein folding reaction by pressure-dependent NMR relaxation dispersion. , 2006, Journal of the American Chemical Society.

[52]  A. Shimizu,et al.  Pressure-tuning FT-IR spectroscopic study on the helix-coil transition of Ala-rich oligopeptide in aqueous solution. , 2005, Biochimica et biophysica acta.

[53]  Shigeyuki Yokoyama,et al.  NMR snapshots of a fluctuating protein structure: ubiquitin at 30 bar-3 kbar. , 2005, Journal of molecular biology.

[54]  A. Gronenborn,et al.  Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR. , 2004, Journal of molecular biology.

[55]  Dmitri I. Svergun,et al.  PRIMUS: a Windows PC-based system for small-angle scattering data analysis , 2003 .

[56]  F. Collart,et al.  A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. , 2002, Protein expression and purification.

[57]  C. Royer.  Revisiting volume changes in pressure-induced protein unfolding. , 2002, Biochimica et biophysica acta.

[58]  S. Garde,et al.  Molecular dynamics simulations of pressure effects on hydrophobic interactions. , 2001, Journal of the American Chemical Society.

[59]  K. Akasaka,et al.  Low-lying excited states of proteins revealed from nonlinear pressure shifts in 1H and 15N NMR. , 2001, Biochemistry.

[60]  C. Royer,et al.  Volume, expansivity and isothermal compressibility changes associated with temperature and pressure unfolding of Staphylococcal nuclease. , 2001, Journal of molecular biology.

[61]  G. Hummer,et al.  The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[62]  G. Dreyfuss,et al.  A Novel Receptor-Mediated Nuclear Protein Import Pathway , 1996, Cell.

[63]  S. Riva,et al.  hnRNP A1 selectively interacts through its Gly-rich domain with different RNA-binding proteins. , 1996, Journal of molecular biology.

[64]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[65]  A. Gronenborn,et al.  A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. , 1993, Science.

[66]  D. Svergun,et al.  Structural analysis of intrinsically disordered proteins by small-angle X-ray scattering. , 2012, Molecular bioSystems.

[67]  G. Wagner,et al.  Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies , 2010, Journal of biomolecular NMR.