scholarly journals Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Baoyu Chen ◽  
Hui-Ting Chou ◽  
Chad A Brautigam ◽  
Wenmin Xing ◽  
Sheng Yang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Biochemical Data ◽  
Actin Assembly ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Regulatory Complex ◽  
Rac1 Gtpase

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

scholarly journals The Relative Binding Position of Nck and Grb2 Adaptors Dramatically Impacts Actin-Based Motility of Vaccinia Virus

2021 ◽  
Author(s):  
Angika Basant ◽  
Michael Way
Keyword(s):  
Vaccinia Virus ◽  
Binding Sites ◽  
Actin Polymerization ◽  
Cellular Processes ◽  
Viral Spread ◽  
Relative Binding ◽  
Signal Output ◽  
Signalling Cascade ◽  
Binding Position

ABSTRACTPhosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to nucleate complex signalling networks. The concept of phase separation has recently changed our appreciation of such multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now explored this parameter in the assembly and operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3-mediated actin polymerization. We generated synthetic networks on Vaccinia by manipulating pTyr motifs in A36 and the unrelated p14 from Orthoreovirus. In contrast to predictions, we find that only specific spatial arrangements of Grb2 and Nck binding sites result in robust N-WASP recruitment, Arp2/3 driven actin polymerization and viral spread. Our results suggest that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are usually conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those that undergo phase transitions.

scholarly journals Structure of CYRI-B (FAM49B), a key regulator of cellular actin assembly

2020 ◽  
Vol 76 (10) ◽  
pp. 1015-1024 ◽  
Author(s):  
Elise Kaplan ◽  
Rachael Stone ◽  
Peter J. Hume ◽  
Nicholas P. Greene ◽  
Vassilis Koronakis
Keyword(s):  
Actin Polymerization ◽  
Small Gtpase ◽  
Actin Assembly ◽  
X Ray ◽  
Rhincodon Typus ◽  
X Ray Crystallography ◽  
Conserved Domain ◽  
Rac1 Activity ◽  
Regulatory Complex ◽  
Lamellipodia Formation

In eukaryotes, numerous fundamental processes are controlled by the WAVE regulatory complex (WRC) that regulates cellular actin polymerization, crucial for cell motility, cell–cell adhesion and epithelial differentiation. Actin assembly is triggered by interaction of the small GTPase Rac1 with CYFIP1, a key component of the WRC. Previously known as FAM49B, CYRI-B is a protein that is highly conserved across the Eukaryota and has recently been revealed to be a key regulator of Rac1 activity. Mutation of CYRI-B or alteration of its expression therefore leads to altered actin nucleation dynamics, with impacts on lamellipodia formation, cell migration and infection by intracellular pathogens. In addition, knockdown of CYRI-B expression in cancer cell lines results in accelerated cell proliferation and invasiveness. Here, the structure of Rhincodon typus (whale shark) CYRI-B is presented, which is the first to be reported of any CYRI family member. Solved by X-ray crystallography, the structure reveals that CYRI-B comprises three distinct α-helical subdomains and is highly structurally related to a conserved domain present in CYFIP proteins. The work presented here establishes a template towards a better understanding of CYRI-B biological function.

scholarly journals Cryo-electron microscopy structure of a nucleosome-bound SWI/SNF chromatin remodeling complex

2019 ◽  
Author(s):  
Yan Han ◽  
Alexis A Reyes ◽  
Sara Malik ◽  
Yuan He
Keyword(s):  
Electron Microscopy ◽  
Chromatin Remodeling ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Gene Activation ◽  
The Body ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Chromatin Remodeling Complex ◽  
High Resolution Structure

AbstractThe multi-subunit chromatin remodeling complex SWI/SNF1–3 is highly conserved from yeast to humans and plays critical roles in various cellular processes including transcription and DNA damage repair4, 5. It uses the energy from ATP hydrolysis to remodel chromatin structure by sliding and evicting the histone octamer6–10, creating DNA regions that become accessible to other essential protein complexes. However, our mechanistic understanding of the chromatin remodeling activity is largely hindered by the lack of a high-resolution structure of any complex from this family. Here we report the first structure of SWI/SNF from the yeast S. cerevisiae bound to a nucleosome at near atomic resolution determined by cryo-electron microscopy (cryo-EM). In the structure, the Arp module is sandwiched between the ATPase and the Body module of the complex, with the Snf2 HSA domain connecting all modules. The HSA domain also extends into the Body and anchors at the opposite side of the complex. The Body contains an assembly scaffold composed of conserved subunits Snf12 (SMARCD/BAF60), Snf5 (SMARCB1/BAF47/ INI1) and an asymmetric dimer of Swi3 (SMARCC/BAF155/170). Another conserved subunit Swi1 (ARID1/BAF250) folds into an Armadillo (ARM) repeat domain that resides in the core of the SWI/SNF Body, acting as a molecular hub. In addition to the interaction between Snf2 and the nucleosome, we also observed interactions between the conserved Snf5 subunit and the histones at the acidic patch, which could serve as an anchor point during active DNA translocation. Our structure allows us to map and rationalize a subset of cancer-related mutations in the human SWI/SNF complex and propose a model of how SWI/SNF recognizes and remodels the +1 nucleosome to generate nucleosome-depleted regions during gene activation11–13.

scholarly journals Structure of the posttranslational Sec protein-translocation channel complex from yeast

Science ◽  
2018 ◽  
Vol 363 (6422) ◽  
pp. 84-87 ◽  
Author(s):  
Samuel Itskanov ◽  
Eunyong Park
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Binding Sites ◽  
Protein Translocation ◽  
Secretory Proteins ◽  
Conducting Channel ◽  
Cryo Electron Microscopy ◽  
Lateral Gate

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

scholarly journals Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno

2020 ◽  
Vol 21 (7) ◽  
pp. 2457 ◽  
Author(s):  
Vikash Singh ◽  
Anthony C. Davidson ◽  
Peter J. Hume ◽  
Vassilis Koronakis
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Nucleotide Exchange Factor ◽  
Regulatory Complex

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.

scholarly journals Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Muscle Actin ◽  
Hydrophobic Cavity ◽  
Cryo Electron Microscopy ◽  
Kinetics Of

AbstractSince the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

scholarly journals PRISM-EM: template interface-based modelling of multi-protein complexes guided by cryo-electron microscopy density maps

2016 ◽  
Vol 72 (10) ◽  
pp. 1137-1148 ◽  
Author(s):  
Guray Kuzu ◽  
Ozlem Keskin ◽  
Ruth Nussinov ◽  
Attila Gursoy
Keyword(s):  
Electron Microscopy ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Protein Assemblies ◽  
New Approach ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Density Maps ◽  
Crystallographic Structures ◽  
Hiv 1

The structures of protein assemblies are important for elucidating cellular processes at the molecular level. Three-dimensional electron microscopy (3DEM) is a powerful method to identify the structures of assemblies, especially those that are challenging to study by crystallography. Here, a new approach, PRISM-EM, is reported to computationally generate plausible structural models using a procedure that combines crystallographic structures and density maps obtained from 3DEM. The predictions are validated against seven available structurally different crystallographic complexes. The models display mean deviations in the backbone of <5 Å. PRISM-EM was further tested on different benchmark sets; the accuracy was evaluated with respect to the structure of the complex, and the correlation with EM density maps and interface predictions were evaluated and compared with those obtained using other methods. PRISM-EM was then used to predict the structure of the ternary complex of the HIV-1 envelope glycoprotein trimer, the ligand CD4 and the neutralizing protein m36.

scholarly journals Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

2018 ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Atp Hydrolysis ◽  
Regulatory Proteins ◽  
Actin Assembly ◽  
Cryo Electron Microscopy ◽  
Pointed End ◽  
Binding Loop

AbstractWe used electron cryo-micrographs to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5’-triphosphate, an ATP analog), ADP-Pi (ADP with inorganic phosphate) or ADP to resolutions of 3.4 Å, 3.4 Å and 3.6 Å. Subunits in the three filaments have nearly identical backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the conformations of the three ATP phosphates and the side chains of Gln137 and His161 in the active site. Flattening also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. Loss of hydrogen bonds after phosphate dissociation may account for the greater flexibility of ADP-actin filaments.Significance StatementActin filaments comprise a major part of the cytoskeleton of eukaryotic cells and serve as tracks for myosin motor proteins. The filaments assemble from actin monomers with a bound ATP. After polymerization, actin rapidly hydrolyzes the bound ATP and slowly dissociates the γ-phosphate. ADP-actin filaments then disassemble to recycle the subunits. Understanding how actin filaments assemble, disassemble and interact with numerous regulatory proteins depends on knowing the structure of the filament. High quality structures of ADP-actin filaments were available, but not of filaments with bound ATP- or with ADP and phosphate. We determined structures of actin filaments with bound AMPPNP (a slowly hydrolyzed ATP analog), ADP and phosphate and ADP by cryo-electron microscopy. These structures show how conformational changes during actin assembly promote ATP hydrolysis and faster growth at one end of the filament than the other.

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