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.

scholarly journals Cryo-EM structure of OSCA1.2 fromOryza sativaelucidates the mechanical basis of potential membrane hyperosmolality gating

2019 ◽  
Vol 116 (28) ◽  
pp. 14309-14318 ◽  
Author(s):  
Koustav Maity ◽  
John M. Heumann ◽  
Aaron P. McGrath ◽  
Noah J. Kopcho ◽  
Po-Kai Hsu ◽  
...  
Keyword(s):  
Conformational Dynamics ◽  
Turgor Pressure ◽  
Environmental Water ◽  
Structural Basis ◽  
Molecular Architecture ◽  
Transport Pathway ◽  
Structural Mechanism ◽  
Calcium Dependent ◽  
Exchange Mass Spectrometry ◽  
And Function

Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality inArabidopsis. Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (Oryza sativa; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially 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 TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.

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 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 basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d

2018 ◽  
Author(s):  
Cheng Zhang ◽  
Silvana Konermann ◽  
Nicholas J. Brideau ◽  
Peter Lotfy ◽  
Scott J. Novick ◽  
...  
Keyword(s):  
Conformational Dynamics ◽  
Enzyme Activation ◽  
Structural Basis ◽  
Binary Complex ◽  
Molecular Architecture ◽  
Guide Rna ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Hydrogen Deuterium ◽  
Target Rna

AbstractCRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function, we resolved cryo-electron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, explain the compact molecular architecture of Cas13d and provide insights into the structural transitions required for enzyme activation. Our comprehensive analysis of Cas13d in diverse enzymatic states facilitated site-specific truncations for minimal size and delineates a blueprint for improving biomolecular applications of RNA targeting.

scholarly journals C2CD6 is required for assembly of the CatSper calcium channel complex and fertilization

2021 ◽  
Author(s):  
Fang Yang ◽  
Maria Gracia Gervasi ◽  
N. Adrian Leu ◽  
Darya A Tourzani ◽  
Gordon Ruthel ◽  
...  
Keyword(s):  
Ion Channel ◽  
Calcium Sensor ◽  
Uncharacterized Protein ◽  
Calcium Dependent ◽  
Heterologous System ◽  
Fertilization Capacity ◽  
And Function ◽  
Essential Subunit

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here we report identification of an uncharacterized protein C2CD6 as a novel subunit of the CatSper ion channel complex. C2CD6 contains a calcium-dependent membrane targeting C2 domain. C2CD6 interacts with the CatSper calcium-selective core forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. Interestingly, transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm in vitro. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 may regulate CatSper assembly, target the CatSper complex to flagellar plasma membrane, and function as a calcium sensor. The identification of C2CD6 as an essential subunit may facilitate the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

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>

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