scholarly journals A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

2015 ◽  
Vol 146 (4) ◽  
pp. 323-340 ◽  
Author(s):  
Nicolaus Schmandt ◽  
Phanindra Velisetty ◽  
Sreevatsa V. Chalamalasetti ◽  
Richard A. Stein ◽  
Ross Bonner ◽  
...  
Keyword(s):  
Ion Channel ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Conformational Dynamics ◽  
Primary Amines ◽  
Molecular Architecture ◽  
X Ray Crystallography ◽  
Gated Channel ◽  
Activation Mechanisms ◽  
Double Electron Electron Resonance

Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.

scholarly journals Cryo-EM Structure of OSCA1.2 from Oryza sativa: Mechanical basis of hyperosmolality-gating in Plants

2018 ◽  
Author(s):  
Koustav Maity ◽  
John Heumann ◽  
Aaron P McGrath ◽  
Noah J Kopcho ◽  
Po-Kai Hsu ◽  
...  
Keyword(s):  
Ion Channel ◽  
Transmembrane Domain ◽  
Conformational Dynamics ◽  
Turgor Pressure ◽  
Environmental Water ◽  
Structural Basis ◽  
Molecular Architecture ◽  
Calcium Dependent ◽  
Lateral Tension ◽  
And Function

Sensing and responding to environmental water deficiencies is essential for the growth, development and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolarity in Arabidopsis. Here, we report the cryo-EM structure and function of an ion channel from rice (Oryza stativa; OsOSCA1.2), showing how it mediates hyperosmolality sensing and ion permeability. The structure reveals a dimer, the molecular architecture of each subunit consists of eleven transmembrane helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The transmembrane domain is structurally related to the TMEM16 family of calcium dependent ion channels and scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms parallel to the plasma membrane and well positioned to sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the transmembrane domain to open the channel and HDX mass spectrometry experimentally confirmed the conformational dynamics of these coupled domains. The structure provides a framework to understand the structural basis of hyperosmolality sensing in crop plants, extending our knowledge of the anoctamin superfamily important for plants and fungi as well as structural mechanisms that can translate membrane stress to ion transporter regulation.

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

2017 ◽  
Author(s):  
Jana Shen ◽  
Zhi Yue ◽  
Helen Zgurskaya ◽  
Wei Chen
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Proton Release ◽  
Intrinsic Resistance ◽  
Constant Ph ◽  
Wide Range ◽  
Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood. <p><br></p>

scholarly journals Structure of the human epithelial sodium channel by cryo-electron microscopy

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sigrid Noreng ◽  
Arpita Bharadwaj ◽  
Richard Posert ◽  
Craig Yoshioka ◽  
Isabelle Baconguis
Keyword(s):  
Electron Microscopy ◽  
Ion Channel ◽  
Sodium Channel ◽  
Conformational Changes ◽  
Single Particle ◽  
Transmembrane Domain ◽  
Epithelial Sodium Channel ◽  
Cryo Electron Microscopy ◽  
Inhibitory Domain ◽  
Epithelial Sodium Channel Enac

The epithelial sodium channel (ENaC), a member of the ENaC/DEG superfamily, regulates Na+ and water homeostasis. ENaCs assemble as heterotrimeric channels that harbor protease-sensitive domains critical for gating the channel. Here, we present the structure of human ENaC in the uncleaved state determined by single-particle cryo-electron microscopy. The ion channel is composed of a large extracellular domain and a narrow transmembrane domain. The structure reveals that ENaC assembles with a 1:1:1 stoichiometry of α:β:γ subunits arranged in a counter-clockwise manner. The shape of each subunit is reminiscent of a hand with key gating domains of a ‘finger’ and a ‘thumb.’ Wedged between these domains is the elusive protease-sensitive inhibitory domain poised to regulate conformational changes of the ‘finger’ and ‘thumb’; thus, the structure provides the first view of the architecture of inhibition of ENaC.

scholarly journals High-Speed AFM directly visualizes conformational dynamics of the HIV-Vif protein complex

2020 ◽  
Author(s):  
Yangang Pan ◽  
Luda S. Shlyakhtenko ◽  
Yuri L. Lyubchenko
Keyword(s):  
Conformational Changes ◽  
Protein Complex ◽  
High Speed ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Time Lapse ◽  
Hiv Treatment ◽  
X Ray Crystallography ◽  
Host Cell Proteins ◽  
Virus Survival

AbstractViral infectivity factor (Vif) is a protein that is essential for the replication of the HIV-1 virus. The key function of Vif is to disrupt the antiviral activity of APOBEC3 proteins, which mutate viral nucleic acids. Inside the cell, Vif binds to the host cell proteins Elongin-C, Elongin-B, and CBF-β, forming a four-protein complex called VCBC. The structure of VCBC in complex with the Cullin5 (Cul5) protein has been solved by X-ray crystallography, and recently, using molecular dynamic (MD) simulations, the dynamics of VCBC and VCBC-Cul5 complexes were characterized. Here, we applied time-lapse high-speed atomic force microscopy (HS-AFM) to visualize the conformational changes of the VCBC complex. We determined the three most favorable conformations of the VCBC complex, which we identified as triangle, dumbbell, and globular structures. In addition, we characterized the dynamics of each of these structures. While our data show a very dynamic behavior for all these structures, we found the triangle and dumbbell structures to be the most dynamic. These findings provide insight into the structure and dynamics of the VCBC complex and support further research into the improvement of HIV treatment, as Vif is essential for virus survival in the cell.

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

2017 ◽  
Author(s):  
Jana Shen ◽  
Zhi Yue ◽  
Helen Zgurskaya ◽  
Wei Chen
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Proton Release ◽  
Intrinsic Resistance ◽  
Constant Ph ◽  
Wide Range ◽  
Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood. <p><br></p>

Quantitative studies of ribosome conformational dynamics

2007 ◽  
Vol 40 (2) ◽  
pp. 163-189 ◽  
Author(s):  
Christopher S. Fraser ◽  
Jennifer A. Doudna
Keyword(s):  
Protein Synthesis ◽  
Single Molecule ◽  
Conformational Changes ◽  
Conformational Dynamics ◽  
Start Codon ◽  
X Ray Crystallography ◽  
Initiation Factors ◽  
Quantitative Studies ◽  
Order Of Magnitude ◽  
Protein Synthesis Initiation

AbstractThe ribosome is a dynamic machine that undergoes many conformational rearrangements during the initiation of protein synthesis. Significant differences exist between the process of protein synthesis initiation in eubacteria and eukaryotes. In particular, the initiation of eukaryotic protein synthesis requires roughly an order of magnitude more initiation factors to promote efficient mRNA recruitment and ribosomal recognition of the start codon than are needed for eubacterial initiation. The mechanisms by which these initiation factors promote ribosome conformational changes during stages of initiation have been studied using cross-linking, footprinting, site-directed probing, cryo-electron microscopy, X-ray crystallography, fluorescence spectroscopy and single-molecule techniques. Here, we review how the results of these different approaches have begun to converge to yield a detailed molecular understanding of the dynamic motions that the eukaryotic ribosome cycles through during the initiation of protein synthesis.

scholarly journals Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening

2011 ◽  
Vol 138 (2) ◽  
pp. 179-194 ◽  
Author(s):  
Iehab Talukder ◽  
Lonnie P. Wollmuth
Keyword(s):  
Nmda Receptor ◽  
Ligand Binding ◽  
Ion Channel ◽  
Nmda Receptors ◽  
Transmembrane Domain ◽  
Conformational Dynamics ◽  
Functional Feature ◽  
Channel Pore ◽  
Activation Gating ◽  
Pore Opening

The defining functional feature of N-methyl-d-aspartate (NMDA) receptors is activation gating, the energetic coupling of ligand binding into opening of the associated ion channel pore. NMDA receptors are obligate heterotetramers typically composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits that gate in a concerted fashion, requiring all four ligands to bind for subsequent opening of the channel pore. In an individual subunit, the extracellular ligand-binding domain, composed of discontinuous polypeptide segments S1 and S2, and the transmembrane channel–forming domain, composed of M1–M4 segments, are connected by three linkers: S1–M1, M3–S2, and S2–M4. To study subunit-specific events during pore opening in NMDA receptors, we impaired activation gating via intrasubunit disulfide bonds connecting the M3–S2 and S2–M4 in either the GluN1 or GluN2A subunit, thereby interfering with the movement of the M3 segment, the major pore-lining and channel-gating element. NMDA receptors with gating impairments in either the GluN1 or GluN2A subunit were dramatically resistant to channel opening, but when they did open, they showed only a single-conductance level indistinguishable from wild type. Importantly, the late gating steps comprising pore opening to its main long-duration open state were equivalently affected regardless of which subunit was constrained. Thus, the NMDA receptor ion channel undergoes a pore-opening mechanism in which the intrasubunit conformational dynamics at the level of the ligand-binding/transmembrane domain (TMD) linkers are tightly coupled across the four subunits. Our results further indicate that conformational freedom of the linkers between the ligand-binding and TMDs is critical to the activation gating process.

scholarly journals Structural titration of receptor ion channel GLIC gating by HS-AFM

2018 ◽  
Vol 115 (41) ◽  
pp. 10333-10338 ◽  
Author(s):  
Yi Ruan ◽  
Kevin Kao ◽  
Solène Lefebvre ◽  
Arin Marchesi ◽  
Pierre-Jean Corringer ◽  
...  
Keyword(s):  
Ion Channel ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
High Speed ◽  
Conformational Dynamics ◽  
Protein Protein Interactions ◽  
Ion Conductance ◽  
Single Molecule Level ◽  
Selective Channel

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein–protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

scholarly journals Shining light on cysteine modification: connecting protein conformational dynamics to catalysis and regulation

2019 ◽  
Vol 26 (4) ◽  
pp. 958-966 ◽  
Author(s):  
Henry van den Bedem ◽  
Mark A Wilson
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Cysteine Residues ◽  
Cysteine Oxidation ◽  
X Ray ◽  
Photo Oxidation ◽  
X Ray Crystallography ◽  
Important Amino Acid

Cysteine is a rare but functionally important amino acid that is often subject to covalent modification. Cysteine oxidation plays an important role in many human disease processes, and basal levels of cysteine oxidation are required for proper cellular function. Because reactive cysteine residues are typically ionized to the thiolate anion (Cys-S−), their formation of a covalent bond alters the electrostatic and steric environment of the active site. X-ray-induced photo-oxidation to sulfenic acids (Cys-SOH) can recapitulate some aspects of the changes that occur under physiological conditions. Here we propose how site-specific cysteine photo-oxidation can be used to interrogate ensuing changes in protein structure and dynamics at atomic resolution. Although this powerful approach can connect cysteine covalent modification to global protein conformational changes and function, careful biochemical validation must accompany all such studies to exclude misleading artifacts. New types of X-ray crystallography experiments and powerful computational methods are creating new opportunities to connect conformational dynamics to catalysis for the large class of systems that use covalently modified cysteine residues for catalysis or regulation.

scholarly journals Cargo modulates the conformation of the nuclear pore in living cells

2020 ◽  
Author(s):  
Joan Pulupa ◽  
Harriet Prior ◽  
Daniel S. Johnson ◽  
Sanford M. Simon
Keyword(s):  
Conformational Changes ◽  
Nuclear Pore Complex ◽  
Conformational Dynamics ◽  
Living Cells ◽  
Nuclear Pore ◽  
Static Structure ◽  
X Ray ◽  
X Ray Crystallography

AbstractWhile the static structure of the nuclear pore complex (NPC) continues to be refined with cryo-EM and x-ray crystallography, the in vivo conformational dynamics of the NPC remain under-explored. We developed sensors that report on the orientation of NPC components by rigidly conjugating mEGFP to different NPC proteins. Our studies show conformational changes to select domains of Nups within the inner ring (Nup54, Nup58, Nup62) when transport through the NPC is perturbed and no conformational changes to Nups elsewhere in the NPC. Our results suggest that select components of the NPC are flexible and undergo conformational changes upon engaging with cargo.

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