scholarly journals Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dawid Deneka ◽  
Sonja Rutz ◽  
Cedric A. J. Hutter ◽  
Markus A. Seeger ◽  
Marta Sawicka ◽  
...  
Keyword(s):  
Molecular Architecture ◽  
Leucine Rich Repeat ◽  
Channel Activity ◽  
Cytoplasmic Domains ◽  
Anion Channels ◽  
Cryo Electron Microscopy ◽  
Allosteric Mechanisms ◽  
Lrr Domain ◽  
Enhance Activity

AbstractMembers of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. Despite their known molecular architecture, the mechanism of activation and the role of the LRR domains in this process has remained elusive. Here we address this question by generating synthetic nanobodies, termed sybodies, which target the LRR domain of the obligatory subunit LRRC8A. We use these binders to investigate their interaction with homomeric LRRC8A channels by cryo-electron microscopy and the consequent effect on channel activation by electrophysiology. The five identified sybodies either inhibit or enhance activity by binding to distinct epitopes of the LRR domain, thereby altering channel conformations. In combination, our work provides a set of specific modulators of LRRC8 proteins and reveals the role of their cytoplasmic domains as regulators of channel activity by allosteric mechanisms.

scholarly journals Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

2018 ◽  
Author(s):  
David M. Kern ◽  
SeCheol Oh ◽  
Richard K. Hite ◽  
Stephen G. Brohawn
Keyword(s):  
Lipid Bilayers ◽  
Critical Role ◽  
Anion Channel ◽  
Lipid Binding ◽  
Channel Structure ◽  
Anion Channels ◽  
Cryo Electron Microscopy ◽  
Lipid Nanodiscs ◽  
Insight Into

AbstractHypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

scholarly journals Molecular basis for the R-type anion channel QUAC1 activity in guard cells

2021 ◽  
Author(s):  
Li Qin ◽  
Ling-hui Tang ◽  
Jia-shu Xu ◽  
Xian-hui Zhang ◽  
Yun Zhu ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Stomatal Closure ◽  
Anion Channel ◽  
Guard Cells ◽  
Channel Activity ◽  
Cytoplasmic Domains ◽  
Anion Channels ◽  
Channel Activation ◽  
Environmental Stimuli ◽  
Functional Analyses

SUMMARYThe rapid (R)-type anion channel plays a central role in controlling stomatal closure in plant guard cells, thus regulating the exchange of water and photosynthetic gas (CO2) in response to environmental stimuli. The activity of the R- type anion channel is regulated by malate. However, the molecular basis of the R-type anion channel activity remains elusive. Here, we describe the first cryo-EM structure of the R-type anion channel QUAC1 at 3.5 Å resolution in the presence of malate. The structure reveals that the QUAC1 is a symmetrical dimer, forming a single electropositive T-shaped pore for passing anions across the membrane. The transmembrane and cytoplasmic domains are assembled into a twisted bi-layer architecture, with the associated dimeric interfaces nearly perpendicular. Our structural and functional analyses reveal that QUAC1 functions as an inward rectifying anion channel and suggests a mechanism for malate-mediated channel activation. Altogether, our study uncovers the molecular basis for a novel class of anion channels and provides insights into the gating and modulation of the R-type anion channel.

scholarly journals Role of the leucine-rich repeat (LRR) domain of cryopyrin/NALP3 in monosodium urate crystal-induced inflammation

2010 ◽  
pp. n/a-n/a ◽  
Author(s):  
Hal M. Hoffman ◽  
Peter Scott ◽  
James L. Mueller ◽  
Amir Misaghi ◽  
Sean Stevens ◽  
...  
Keyword(s):  
Leucine Rich Repeat ◽  
Monosodium Urate ◽  
Urate Crystal ◽  
Lrr Domain

scholarly journals Cryo-EM structures of the TTYH family reveal a novel architecture for lipid interactions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anastasiia Sukalskaia ◽  
Monique S. Straub ◽  
Dawid Deneka ◽  
Marta Sawicka ◽  
Raimund Dutzler
Keyword(s):  
Anion Channel ◽  
Extracellular Domain ◽  
Cell Swelling ◽  
Anion Channels ◽  
Lipid Interactions ◽  
Transmembrane Segments ◽  
Cryo Electron Microscopy ◽  
The Brain ◽  
Eukaryotic Membrane Proteins

AbstractThe Tweety homologs (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain. The three human paralogs were assigned to function as anion channels that are either activated by Ca2+ or cell swelling. To uncover their unknown architecture and its relationship to function, we have determined the structures of human TTYH1–3 by cryo-electron microscopy. All structures display equivalent features of a dimeric membrane protein that contains five transmembrane segments and an extended extracellular domain. As none of the proteins shows attributes reminiscent of an anion channel, we revisited functional experiments and did not find any indication of ion conduction. Instead, we find density in an extended hydrophobic pocket contained in the extracellular domain that emerges from the lipid bilayer, which suggests a role of TTYH proteins in the interaction with lipid-like compounds residing in the membrane.

scholarly journals F-Box Protein Grr1 Interacts with Phosphorylated Targets via the Cationic Surface of Its Leucine-Rich Repeat

2001 ◽  
Vol 21 (7) ◽  
pp. 2506-2520 ◽  
Author(s):  
Yuchu G. Hsiung ◽  
Hui-Chu Chang ◽  
Jean-Luc Pellequer ◽  
Roberto La Valle ◽  
Stefan Lanker ◽  
...  
Keyword(s):  
Structural Model ◽  
Positive Charge ◽  
Convex Surface ◽  
Point Mutations ◽  
Concave Surface ◽  
Leucine Rich Repeat ◽  
Carboxy Terminus ◽  
F Box Protein ◽  
Lrr Domain

ABSTRACT The flexibility and specificity of ubiquitin-dependent proteolysis are mediated, in part, by the E3 ubiquitin ligases. One class of E3 enzymes, SKp1/cullin/F-box protein (SCF), derives its specificity from F-box proteins, a heterogeneous family of adapters for target protein recognition. Grr1, the F-box component of SCFGrr1, mediates the interaction with phosphorylated forms of the G1 cyclins Cln1 and Cln2. We show that binding of Cln2 by SCFGrr1 was dependent upon its leucine-rich repeat (LRR) domain and its carboxy terminus. Our structural model for the Grr1 LRR predicted a high density of positive charge on the concave surface of the characteristic horseshoe structure. We hypothesized that specific basic residues on the predicted concave surface are important for recognition of phosphorylated Cln2. We show that point mutations that converted the basic residues on the concave surface but not those on the convex surface to neutral or acidic residues interfered with the capacity of Grr1 to bind to Cln2. The same mutations resulted in the stabilization of Cln2 and Gic2 and also in a spectrum of phenotypes characteristic of inactivation of GRR1, including hyperpolarization and enhancement of pseudohyphal growth. It was surprising that the same residues were not important for the role of Grr1 in nutrient-regulated transcription of HXT1 or AGP1. We concluded that the cationic nature of the concave surface of the Grr1 LRR is critical for the recognition of phosphorylated targets of SCFGrr1 but that other properties of Grr1 are required for its other functions.

scholarly journals Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
David M Kern ◽  
SeCheol Oh ◽  
Richard K Hite ◽  
Stephen G Brohawn
Keyword(s):  
Lipid Bilayers ◽  
Critical Role ◽  
Anion Channel ◽  
Lipid Binding ◽  
Channel Structure ◽  
Anion Channels ◽  
Cryo Electron Microscopy ◽  
Lipid Nanodiscs ◽  
Insight Into

Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here, we present single-particle cryo-electron microscopy structures of Mus musculus LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

scholarly journals Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Sensen Zhang ◽  
Baolei Yuan ◽  
Jordy Homing Lam ◽  
Jun Zhou ◽  
Xuan Zhou ◽  
...  
Keyword(s):  
Md Simulation ◽  
Potential Role ◽  
Md Simulations ◽  
Full Length ◽  
Inflammasome Activation ◽  
Simulation Studies ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Atp Efflux

AbstractPannexin1 (PANX1) is a large-pore ATP efflux channel with a broad distribution, which allows the exchange of molecules and ions smaller than 1 kDa between the cytoplasm and extracellular space. In this study, we show that in human macrophages PANX1 expression is upregulated by diverse stimuli that promote pyroptosis, which is reminiscent of the previously reported lipopolysaccharide-induced upregulation of PANX1 during inflammasome activation. To further elucidate the function of PANX1, we propose the full-length human Pannexin1 (hPANX1) model through cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulation studies, establishing hPANX1 as a homo-heptamer and revealing that both the N-termini and C-termini protrude deeply into the channel pore funnel. MD simulations also elucidate key energetic features governing the channel that lay a foundation to understand the channel gating mechanism. Structural analyses, functional characterizations, and computational studies support the current hPANX1-MD model, suggesting the potential role of hPANX1 in pyroptosis during immune responses.

scholarly journals The mycoplasma surface proteins MIB and MIP promote the dissociation of the antibody-antigen interaction

2021 ◽  
Vol 7 (10) ◽  
pp. eabf2403
Author(s):  
Pierre Nottelet ◽  
Laure Bataille ◽  
Geraldine Gourgues ◽  
Robin Anger ◽  
Carole Lartigue ◽  
...  
Keyword(s):  
Surface Proteins ◽  
Mycoplasma Species ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Fab Fragment ◽  
Cryo Electron Microscopy ◽  
Antigen Complex ◽  
Antigen Interaction ◽  
Immunoglobulin Binding

Mycoplasma immunoglobulin binding (MIB) and mycoplasma immunoglobulin protease (MIP) are surface proteins found in the majority of mycoplasma species, acting sequentially to capture antibodies and cleave off their VH domains. Cryo–electron microscopy structures show how MIB and MIP bind to a Fab fragment in a “hug of death” mechanism. As a result, the orientation of the VL and VH domains is twisted out of alignment, disrupting the antigen binding site. We also show that MIB-MIP has the ability to promote the dissociation of the antibody-antigen complex. This system is functional in cells and protects mycoplasmas from antibody-mediated agglutination. These results highlight the key role of the MIB-MIP system in immunity evasion by mycoplasmas through an unprecedented mechanism, and open exciting perspectives to use these proteins as potential tools in the antibody field.

scholarly journals Novel role of endothelial BKCa channels in altered vasoreactivity following hypoxia

2010 ◽  
Vol 299 (5) ◽  
pp. H1439-H1450 ◽  
Author(s):  
Jennifer M. Hughes ◽  
Melissa A. Riddle ◽  
Michael L. Paffett ◽  
Laura V. Gonzalez Bosc ◽  
Benjimen R. Walker
Keyword(s):  
Ex Vivo ◽  
Barometric Pressure ◽  
Channel Blockers ◽  
Channel Activity ◽  
Resistance Arteries ◽  
Bkca Channels ◽  
Oxide Synthase ◽  
Increased Activity ◽  
Small Conductance

The systemic vasculature exhibits attenuated vasoconstriction following hypobaric chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased activity of endothelial cell (EC) large-conductance, calcium-activated potassium (BKCa) channels contributes to this response. Gracilis resistance arteries from hypobaric CH (barometric pressure = 380 mmHg for 48 h) rats demonstrated reduced myogenic reactivity and hyperpolarized VSM membrane potential ( Em) compared with controls under normoxic ex vivo conditions. These differences were eliminated by endothelial disruption. In the presence of cyclooxygenase and nitric oxide synthase inhibition, combined intraluminal administration of the intermediate and small-conductance, calcium-activated K+ channel blockers TRAM-34 and apamin was without effect on myogenic responsiveness and VSM Em in both groups; however, these variables were normalized in CH arteries by intraluminal administration of the BKCa inhibitor iberiotoxin (IBTX). Basal EC Em was hyperpolarized in arteries from CH rats compared with controls and was restored by IBTX, but not by TRAM-34/apamin. K+ channel blockers were without effect on EC basal Em in controls. Similarly, IBTX blocked acetylcholine-induced dilation in arteries from CH rats, but was without effect in controls, whereas TRAM-34/apamin eliminated dilation in controls. Acetylcholine-induced EC hyperpolarization and calcium responses were inhibited by IBTX in CH arteries and by TRAM-34/apamin in controls. Patch-clamp experiments on freshly isolated ECs demonstrated greater K+ current in cells from CH rats that was normalized by IBTX. IBTX was without effect on K+ current in controls. We conclude that hypobaric CH induces increased endothelial BKCa channel activity that contributes to reduced myogenic responsiveness and EC and VSM cell hyperpolarization.

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