scholarly journals BAR scaffolds drive membrane fission by crowding disordered domains

2018 ◽  
Vol 218 (2) ◽  
pp. 664-682 ◽  
Author(s):  
Wilton T. Snead ◽  
Wade F. Zeno ◽  
Grace Kago ◽  
Ryan W. Perkins ◽  
J Blair Richter ◽  
...  
Keyword(s):  
Bar Domain ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Membrane Fission ◽  
Membrane Surfaces ◽  
Cell Assays ◽  
Key Driver ◽  
Bar Domains ◽  
Lipid Tubules

Cellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysin-rvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of isolated BAR domains in vitro, the current paradigm is that BAR domain–containing proteins polymerize into cylindrical scaffolds that stabilize lipid tubules. But in nature, proteins that contain BAR domains often also contain large intrinsically disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain–containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data suggest that the ability to concentrate disordered domains is a key driver of membrane remodeling and fission by BAR domain–containing proteins.

scholarly journals BAR scaffolds drive membrane fission by crowding disordered domains

2018 ◽  
Author(s):  
Wilton T. Snead ◽  
Wade F. Zeno ◽  
Grace Kago ◽  
Ryan W. Perkins ◽  
J Blair Richter ◽  
...  
Keyword(s):  
Cellular Membranes ◽  
Bar Domain ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Membrane Fission ◽  
Cell Assays ◽  
Membrane Tubules ◽  
Bar Domains ◽  
Lipid Tubules

SummaryCylindrical protein scaffolds are thought to stabilize membrane tubules, preventing membrane fission. In contrast, Snead et al. find that when scaffold proteins assemble, bulky disordered domains within them become acutely concentrated, generating steric pressure that destabilizes tubules, driving fission.AbstractCellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysinrvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of BAR domains in isolation, the current paradigm is that they polymerize into cylindrical scaffolds that stabilize lipid tubules, preventing membrane fission. But in nature BAR domains are often part of multi-domain proteins that contain large intrinsically-disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data challenge the idea that cellular membranes adopt the curvature of BAR scaffolds, suggesting instead that the ability to concentrate disordered domains is the key requirement for membrane remodeling and fission by BAR domain-containing proteins.

scholarly journals Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vikas A. Tillu ◽  
James Rae ◽  
Ya Gao ◽  
Nicholas Ariotti ◽  
Matthias Floetenmeyer ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Membrane Lipid ◽  
Membrane Curvature ◽  
Caveolin 1 ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Solution Generation ◽  
Endosomal System ◽  
Disordered Regions

AbstractCaveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction.

2021 ◽  
Author(s):  
Margaux R. Audett ◽  
Erin L. Johnson ◽  
Jessica M. McGory ◽  
Dylan M. Barcelos ◽  
Evelin Oroszne Szalai ◽  
...  
Keyword(s):  
Protein Binding ◽  
Protein Phosphatase ◽  
Spindle Assembly Checkpoint ◽  
Protein Phosphatase 1 ◽  
Intrinsically Disordered ◽  
Regulatory Circuit ◽  
Cell Assays ◽  
Kinetics Of

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of D. melanogaster KNL1 (Spc105) has never been shown to bind MTs nor to recruit PP1. Furthermore, the phospho-regulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell based-assays. A phospho-regulatory circuit, which utilizes Aurora B kinase (ABK), promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag, and deletion/chimera mutants are used to define the interplay of MT- and PP1-binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

scholarly journals A new class of disordered elements controls DNA replication through initiator self-assembly

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Matthew W Parker ◽  
Maren Bell ◽  
Mustafa Mir ◽  
Jonchee A Kao ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Dna Replication ◽  
Self Assembly ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Linear Arrangement ◽  
New Class ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Liquid Liquid Phase Separation

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

scholarly journals Comparative study of curvature sensing mediated by F-BAR domain and an intrinsically disordered region of FBP17

2020 ◽  
Author(s):  
Maohan Su ◽  
Yinyin Zhuang ◽  
Xinwen Miao ◽  
Yongpeng Zeng ◽  
Weibo Gao ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Membrane Trafficking ◽  
Membrane Curvature ◽  
Wave System ◽  
Common Belief ◽  
Bar Domain ◽  
Curvature Sensing ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Region

Membrane curvature has emerged as an intriguing physical organization principle underlying biological signaling and membrane trafficking. FBP17 of the CIP4/FBP17/Toca-1 F-BAR family is unique in the BAR family because its structurally folded F-BAR domain does not contain any hydrophobic motifs that insert into lipid bilayer. While it has been widely assumed so, whether the banana-shaped F-BAR domain alone can sense curvature has never been experimentally demonstrated. Using a nanopillar-supported lipid bilayer system, we found that the F-BAR domain of FBP17 displayed minimal curvature sensing in vitro. We further identified an alternatively spliced intrinsically disordered region (IDR) of FBP17 next to its F-BAR domain that is conserved in sequence across species. The IDR senses membrane curvature and its sensing ability greatly exceeds that of F-BAR domain alone. In living cells, presence of the IDR domain changed the dynamics of FBP17 recruitment in a curvature-coupled cortical wave system. Collectively, we propose that FBP17 does sense curvature but contrary to the common belief, its curvature sensing capability largely originates from its disordered region, not F-BAR domain itself.

scholarly journals Conserved Outer Tegument Component UL11 from Herpes Simplex Virus 1 Is an Intrinsically Disordered, RNA-Binding Protein

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Claire M. Metrick ◽  
Andrea L. Koenigsberg ◽  
Ekaterina E. Heldwein
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Intrinsic Disorder ◽  
Herpes Simplex Virus 1 ◽  
Tegument Proteins ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT A distinguishing morphological feature of all herpesviruses is the multiprotein tegument layer located between the nucleocapsid and lipid envelope of the virion. Tegument proteins play multiple roles in viral replication, including viral assembly, but we do not yet understand their individual functions or how the tegument is assembled and organized. UL11, the smallest tegument protein, is important for several distinct processes in replication, including efficient virion morphogenesis and cell-cell spread. However, the mechanistic understanding of its role in these and other processes is limited in part by the scant knowledge of its biochemical and structural properties. Here, we report that UL11 from herpes simplex virus 1 (HSV-1) is an intrinsically disordered, conformationally dynamic protein that undergoes liquid-liquid phase separation (LLPS) in vitro. Intrinsic disorder may underlie the ability of UL11 to exert multiple functions and bind multiple partners. Sequence analysis suggests that not only all UL11 homologs but also all HSV-1 tegument proteins contain intrinsically disordered regions of different lengths. The presence of intrinsic disorder, and potentially, the ability to form LLPS, may thus be a common feature of the tegument proteins. We hypothesize that tegument assembly may involve the formation of a biomolecular condensate, driven by the heterogeneous mixture of intrinsically disordered tegument proteins. IMPORTANCE Herpesvirus virions contain a unique tegument layer sandwiched between the capsid and lipid envelope and composed of multiple copies of about two dozen viral proteins. However, little is known about the structure of the tegument or how it is assembled. Here, we show that a conserved tegument protein UL11 from herpes simplex virus 1, a prototypical alphaherpesvirus, is an intrinsically disordered protein that undergoes liquid-liquid phase separation in vitro. Through sequence analysis, we find intrinsically disordered regions of different lengths in all HSV-1 tegument proteins. We hypothesize that intrinsic disorder is a common characteristic of tegument proteins and propose a new model of tegument as a biomolecular condensate.

scholarly journals An in vitro reconstituted U1 snRNP allows the study of the disordered regions of the particle and the interactions with proteins and ligands

2021 ◽  
Author(s):  
Sébastien Campagne ◽  
Tebbe de Vries ◽  
Florian Malard ◽  
Pavel Afanasyev ◽  
Georg Dorn ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Molecular Mechanisms ◽  
Low Complexity ◽  
Stem Loop ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
U1 Snrnp ◽  
Splicing Regulator ◽  
Disordered Regions

Abstract U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon–intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.

scholarly journals Molecular and Biochemical Techniques for Deciphering p53-MDM2 Regulatory Mechanisms

Biomolecules ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 36
Author(s):  
Konstantinos Karakostis ◽  
Ignacio López ◽  
Ana M. Peña-Balderas ◽  
Robin Fåhareus ◽  
Vanesa Olivares-Illana
Keyword(s):  
Protein Complexes ◽  
Post Translational Modifications ◽  
Intrinsically Disordered ◽  
Advantages And Disadvantages ◽  
Intrinsically Disordered Regions ◽  
Multiple Partners ◽  
Allosteric Interactions ◽  
Spatio Temporal

The p53 and Mouse double minute 2 (MDM2) proteins are hubs in extensive networks of interactions with multiple partners and functions. Intrinsically disordered regions help to adopt function-specific structural conformations in response to ligand binding and post-translational modifications. Different techniques have been used to dissect interactions of the p53-MDM2 pathway, in vitro, in vivo, and in situ each having its own advantages and disadvantages. This review uses the p53-MDM2 to show how different techniques can be employed, illustrating how a combination of in vitro and in vivo techniques is highly recommended to study the spatio-temporal location and dynamics of interactions, and to address their regulation mechanisms and functions. By using well-established techniques in combination with more recent advances, it is possible to rapidly decipher complex mechanisms, such as the p53 regulatory pathway, and to demonstrate how protein and nucleotide ligands in combination with post-translational modifications, result in inter-allosteric and intra-allosteric interactions that govern the activity of the protein complexes and their specific roles in oncogenesis. This promotes elegant therapeutic strategies that exploit protein dynamics to target specific interactions.

scholarly journals Distinct RNA polymerase transcripts direct the assembly of phase-separated DBC1 nuclear bodies in different cell lines

2021 ◽  
pp. mbc.E21-02-0081
Author(s):  
Taro Mannen ◽  
Masato Goto ◽  
Takuya Yoshizawa ◽  
Akio Yamashita ◽  
Tetsuro Hirose ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Binding Domains ◽  
Additional Protein ◽  
Protein Components

The mammalian cell nucleus is a highly organized organelle that contains membrane-less structures referred to as nuclear bodies (NBs). Some NBs carry specific RNA types that play architectural roles in their formation. Here, we show two types of RNase-sensitive DBC1-containing NBs: DBC1 nuclear body (DNB) in HCT116 cells and Sam68 nuclear body (SNB) in HeLa cells that exhibit phase-separated features and are constructed using RNA polymerase I or II transcripts in a cell type-specific manner. We identified additional protein components present in DNB by immunoprecipitation-mass spectrometry, some of which (DBC1 and HNRNPL) are required for DNB formation. The rescue experiment using the truncated HNRNPL mutants revealed that two RNA-binding domains and intrinsically disordered regions of HNRNPL play significant roles in DNB formation. All these domains of HNRNPL promote in vitro droplet formation, suggesting the need for multivalent interactions between HNRNPL and RNA as well as proteins in DNB formation.

scholarly journals Defining binding motifs and dynamics of the multi-pocket FERM domain from ezrin, radixin, moesin and merlin

2020 ◽  
Author(s):  
Muhammad Ali ◽  
Alisa Khramushin ◽  
Vikash K Yadav ◽  
Ora Schueler-Furman ◽  
Ylva Ivarsson
Keyword(s):  
Conformational Changes ◽  
In Silico ◽  
Cell Cortex ◽  
List Type ◽  
Binding Assays ◽  
Ferm Domain ◽  
Binding Motifs ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

AbstractThe ERMs (ezrin, radixin and moesin) and the closely related merlin (NF2) participate in signaling events at the cell cortex through interactions mediated by their conserved FERM domain. We systematically investigated the FERM domain mediated interactions with short linear motifs (SLiMs) by screening the FERM domains againsts a phage peptidome representing intrinsically disordered regions of the human proteome. We uncovered a diverse set of interacting partners with similar but distinct binding motifs (FYDF, xYxV, FY(D/E)L and LQE(I/L) that bind to distinct binding pockets. We validated interactions between moesin and merlin FERM domains and full-length FAM83G, HIF1A, LATS1, NOP53, PAK6, RRBP1 and ZNF622 through pull-down experiments. Using biophysical binding assays, we determined affinities of, and uncovered allosteric interdependencies between, different binding partners, suggesting that the FERM domain acts as a switchable interaction hub. Using Rosetta FlexPepDock computational peptide docking protocols, we investigated the energy landscapes of identified interactions, which provide a detailed molecular understanding of the binding of the distinct binding motifs, as well as possible allosteric interconnections. This study demonstrates how experimental and computational approaches together can unravel a complex system of protein-peptide interactions that includes a family of proteins with multiple binding sites that interact with similar but distinct binding motifs.HighlightsWe screened the human disorderome for motif-containing partners of the FERM domainsWe expand the ERM and merlin interactomes of the ERMs and merlinWe identify four distinct motif classes that bind the ERM and merlin FERM domains: FYDF, xYxV, FY(D/E)L and LQE(I/L)In-vitro and in-silico data suggest that the FYDF motif binds to the F3a site and that xYxV motif binds to the F3b siteIn-silico modelling sheds light on the underlying conformational changes responsible for ligand interdependenciesAbstract Figure

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