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July 2017: Evolution of IDPs

Each month the IDP State Letter brings you summaries of recent papers and news in the field of intrinsically disordered proteins. To subscribe to the newsletter, please follow this link and add your name to our mailing list. We thank you for your subscription to the newsletter and welcome you to direct any comments, questions, and concerns to us at IDPStateLetter@gmail.com.

The study of intrinsically disordered proteins and regions (IDRs) has involved work at the intersection of cell biology, biochemistry, in vitro and computational biophysics, and bioinformatics. Over the last thirty years these studies have provided an ever increasingly sophisticated description of protein disorder, and how that disorder is harnessed to drive cellular function.

This month we present a potpourri of papers covering IDP science for you to enjoy. Two papers on liquid-liquid phase separation are presented. The first paper employs advanced polymer physics to recapitulate a rather peculiar case of IDP phase separation by Wei et al. The second work demonstrates the role of membrane-less phase separated organelles in viral replication by Nikolic et al. Three other papers present work on IDPs relevant in various human diseases. Robotta et al demonstrates the effect of Parkinson's disease mutations on the α-synuclein-lipid interaction, Kang et al uses all-atom MD simulation to decode the molecular basis of polyQ aggregation in Huntington’s disease, and Neira et al report explore the IDP Nuclear Protein 1 as a cancer drug target. Enjoy!

Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles

 

Membrane-less organelles represent heterogeneous assemblies of protein and RNA and are believed to perform a wide range of cellular functions. In many cases, their assembly is well described by the physics of liquid-liquid phase separation, and through extensive genetic and biophysical analyses it has become clear that proteins which contain disordered regions appear critical for the formation of these biomolecular condensates. Despite their prevalence, the extract role of disorder in the formation of these assemblies has remained elusive. In this work, Wei et al. explore the biophysical and rheological properties of liquid-droplets formed by the P-granule protein LAF-1 and its RGG domain, a disordered region that is necessary and sufficient for droplet formation.

Using a novel ultra-fast scanning FCS approach, the authors make the surprising discovery that the concentration of protein within LAF-1/RGG droplets formed in vitro is significantly lower than expected, based on conventional ideas and simple mean-field theories. The authors are able to explain this low concentration by fitting their data to an advanced analytical framework that explicitly takes the extent of conformational fluctuations associated with the monomer into consideration. This theory makes quantitative predictions regarding the global dimensions of monomer’s fluctuations and the expected rheological properties of the assemblies. These predictions are verified using simulations, rheological analysis, and probe-accessibility experiments. Several of these experiments were then performed in vivo and with alternative droplet forming proteins, yielding quantitatively similar results. Taken together, these results suggest that for some membrane-less organelles, semi-dilute liquid-scaffolds can emerge and provide a relatively dilute environment, which may be well suited for complex biochemistry and the accommodation of other large macromolecules.  

Rohit Pappu is a professor of biomedical engineering and the director of the center for biological systems engineering at Washington University in St. Louis

Cliff Brangwynne is an associate professor in chemical and biological engineering at Princeton University.

Negri bodies are viral factories with properties of liquid organelles

Negri bodies (NBs) are membraneless cytoplasmic inclusions induced by rabies virus (RABV). It has been shown previously that viral transcription and replication occur in NBs, which contain all the replication machinery, and are very similar to stress granules which are membraneless organelles formed by protein and RNA assemblies. In this work, the authors characterized the physical properties of NBs and confirmed that they are indeed liquid organelles via confocal imaging and FRAP experiments. Furthermore, they discovered that a ribonucleoprotein complex formed by nucleocapsid, RNA polymerase, and its cofactor phosphoprotein, which is important to viral replication, was ejected from NBs, and transported in a microtubule-dependent manner. The authors further characterized the minimal system to form NBs and found that while coexpression of nucleoprotein in the nucleocapsid and the phosphoprotein leads to phase-separated liquid organelles, the amino terminal part of the second intrinsically disordered domain (IDD2) in phosphoprotein is key to the formation of NBs. This work demonstrate the importance of phase separation induced by intrinsically disordered domains in a virus, to allow both the specific recruitment of machineries necessary for viral replication and escape from the cellular antiviral responses.

Yves Gaudin is a group leader at the Institut de Biologie Intégrative de la Cellule (I2BC) 

Identification of a drug targeting an intrinsically disordered protein in pancreatic adenocarcinoma

Nuclear Protein 1 (NUPR1) is an IDP whose specialty is interacting with a number of binding partners related to apoptosis, cell cycle regulation, chromatin remodeling, and TGFβ activity regulation. Indeed, aberrant over-expression is linked to multiple types of cancer. While NUPR1 lacks regular secondary structure, small pockets of residues within its sequence can loosely-associate with various small molecules. Armed with this knowledge, Neira and colleagues set out to investigate whether NUPR1 is a druggable target for pancreatic ductal adenocarcinoma (PDAC). The potential ligands were selected from an array of 1120 FDA-approved drugs, based on whether their presence altered the thermal stability of NUPR1. After the initial screening round, candidates were narrowed down via interrogation of binding activity using a suite of biophysical techniques including ITC, fluorescence dye binding, and NMR. DOSY-NMR guided MD simulations independently verified the residue-specific binding locations, revealing a multi-region “hot-spot” for the most active compound,Trifluoperazine. The effectiveness and specificity of Trifluoperazine to target NUPR1 activity was demonstrated using an array of cell assays, and an in vivo NMRI-Foxn1nu/Foxn1nu mouse model/MiaPaCa-2 tumor graft treatment study. Promisingly, there was a significant dosage-dependent effect on shrinking tumor size. Indeed, while IDPs are non-traditional small molecule drug targets, Neira, et. al show that more effort could indeed be placed into finding these interacting partners, for not only cancer, but for a  growing number of different IDP-associated maladies.

Olga Abian Franco is a Researcher (Miguel Servet program) in the Aragon Health Sciences Institute (IACS)

Alpha-Synuclein Disease Mutations Are Structurally Defective and Locally Affect Membrane Binding

One of the most well-studied IDPs, α-Synuclein is implicated in the pathogenesis of Parkinson’s disease.  This IDP interacts with lipid surfaces and adopts a wide range of conformations. Several mutants of α-Synuclein are associated with Parkinson’s disease and are often believed to have diminished membrane binding affinity.  However, the molecular basis for differential membrane binding is unknown. The authors used continuous wave EPR spectroscopy coupled with site selective spin labelling to assess local membrane binding affinity at multiple sites across the sequence. While the authors found the overall pattern of lipid binding across the sequence to be preserved in the mutants, the mutant affinities were reduced compared to wild-type, with particularly large decreases in affinity at sites close to the mutations. The authors performed the study with model lipids as well as a lipid mixture that mimics the inner mitochondrial membrane and the results were found to be qualitatively similar. This study underscores the need of probing local residue-specific effects to attain a comprehensive perspective on IDP function and binding affinities.

Vinod Subramaniam is Rector Magnificus of the Vrije Universiteit Amsterdam

Malte Drescher is a professor in chemistry at the Universitat Konstanz

Emerging β-Sheet Rich Conformations in Supercompact Huntingtin Exon-1 Mutant Structures

Huntington’s disease (HD) is one of the prominent trinucleotide repeat disorders expansion of nucleotide CAG repeats in exon-1 of the HD gene, leads to an abnormally elongated polyglutamine tract (polyQ) in Huntingtin (Htt) protein leading to increased aggregation and fibrillation propensity. Physiologically only Htt bearing polyQ tracts longer than 36 residues are associated with onset of HD. However how longer and longer polyQ tracts encodes for HD pathogenesis from a molecular perspective and is a matter of intense study. polyQ tracts are disordered and are thus not amenable to conventional structural biology tools and also not easily accessed experimentally owing to very strong aggregation propensity. Thus MD simulations becomes a handy tool to explore such systems with atomistic details. The authors use temperature replica exchange molecular dynamics (T-REMD) simulations to enhance sampling. They observe increasing degree of collapse with polyQ length with very low scaling exponents. They also find a higher propensity of β-sheet type contacts in the ensembles as the polyQ length increases. MD simulations also show that glutamine sidechains pack inside the collapsed polyQ globules thus explaining their very low solubility. Overall the study summarizes a comprehensive picture of the molecular features of polyQ proteins as a function of polyQ length which however might needs validation by experiments and parallel simulation strategies owing to issues such as force-field dependant collapse behaviour of IDPs as had been seen before.

Ruhong Zhou is a Distinguished Research Staff Scientist (DRSM) and Head of Soft Matter Science Dept, at IBM Research, as well as an Adjunct Professor at Department of Chemistry, Columbia University.
IDP State Letter contributors:
Aritra Chowdhury is a 3rd year graduate student in Edward Lemke’s lab at EMBL, Heidelberg. He uses a set of high resolution fluorescence spectroscopy tools, to understand dynamics and binding mechanisms of IDPs and the interplay between the two, with a particular focus on FG nucleoporins.
Xiaohan Li has just graduated from Prof. Elizabeth Rhoades' lab at the University of Pennsylvania trying to elucidate the mechanism of interaction between tau protein and tubulin using single molecule techniques. He is fascinated by the diversity of functions of IDPs and their importance in changing our understanding of structural-to-function diagram for proteins. He will be joining Dr. Madan Babu's lab in LMB in Cambridge UK studying systems biology of IDPs. 
Alex S. Holehouse has just graduated from the Pappu lab at Washington University in St. Louis. He uses a mixture of simulation and theory to ask general, multiscale questions about IDPs. His work focuses on understand the relationship between sequence properties and IDP conformational and collective behaviour, with a particular interest in the sequence determinants of phase separation. He will continue postdocing in the Pappu lab and turning his focus to polymer physics of biological phase separation, the relationship between self-assembly and function, and understanding evolution in IDPs.
Kendra L. Hailey is a post-doctoral research associate in the Patricia Jennings lab at the University of California, San Diego. Her primary area of research relates to examining how protein folding pathways contribute to native protein function and finding novel allosteric regulation mechanisms within a diverse array of systems, especially for the IL-1 family of ligands/receptors.
Jamie is a postdoctoral researcher at the Center for Aerosol Impacts for Climate and the Environment. She uses computational biophysics techniques to understand the dynamic structure of biological molecules within aerosol particles. Her graduate work focused on protein dynamics and especially intrinsically disordered proteins in protein-protein interactions.

What paper do you want to nominate for review? We welcome all readers to nominate papers by sending an email to bps.idp@gmail.com.

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Artwork was contributed by Jamie M. Schiffer, editor of the IDP State Letter, and postdoctoral researcher at UC San Diego. 
 
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