Molecular architecture of the flagellar export apparatus reveals membrane remodeling and conformational changes crucial for flagellar assembly

To highlight the mechanisms of artificial surface/protein/platelet interactions, results obtained by various methods have been integrated to elucidate some of the correlations between phenomena which occur at the macromolecular level and subsequently influence those at the cellular level, such as platelet adhesion. Microcinematographic evidence obtained under the controlled conditions of the Stagnation Point Flow Experiment (SPFE) indicate that, even on glass, platelet adhesion commences only after 30-60 sees of exposure to native blood. This lag period is consistent with diffusion kinetics predicting the arrival of plasma proteins should overhwelmingly precede that of the cellular components. During the lag period, native plasma proteins collide with the artificial surface and, in most cases, adsorb with surface-induced conformational changes. The energy for altering the secondary protein structure is supplied by the heat of adsorption. The extent of adsorption and structural alterations depend upon both the type of protein and the molecular architecture of the artificial surface, viz., the number density and orientation of polar, H-bonding, etc. groups accessible to proteins. Using microparticulate glass (< μ dia.) and a microcalorimeter sensitive to ±0.00001° C in 100 ml of sample volume, serum albumin was found to adsorb, release heat, and desorb in a conformationally altered state. In contrast, γ-(7S)-globulin and fibrinogen underwent irreversible multilayer attachment releasing (1.0-1.7) χ 103 Kcal/mole of protein adsorbed directly to the glass surface. Proteins in the second, etc. sorbed layers released much smaller heats. The electrophoretic mobility of the same particles coated with varying amounts of the same proteins confirmed that the relatively greatest conformational change occurred in the protein layer directly attached to the artificial surface. On homologous Nylons exposed under identical hemodynamic conditions in the SPFE, the surface number density of platelets remaining adherent at points of identical shear was proportionate to the polar force contribution of those surfaces. These results indicate that the protein layer which settles first, is acting as a “proportional transformer” mediating the effects of artificial surfaces onto platelets.

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Mechanisms of activation and desensitization of full-length glycine receptor in membranes

10.1101/788695 ◽

2019 ◽

Cited By ~ 3

Author(s):

Arvind Kumar ◽

Sandip Basak ◽

Shanlin Rao ◽

Yvonne Gicheru ◽

Megan L. Mayer ◽

...

Keyword(s):

Conformational Changes ◽

Single Channel ◽

Glycine Receptor ◽

Full Length ◽

Structural Basis ◽

Molecular Architecture ◽

Channel Conductance ◽

Single Channel Conductance ◽

The Central Nervous System ◽

Dynamics Simulations

AbstractGlycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyR) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structural studies have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unknown. Here, we present Cryo-EM structures of the full-length GlyR protein reconstituted into lipid nanodiscs that are captured in the unliganded (closed) and glycine-bound (open and desensitized) conformations. A comparison of the three states reveals global conformational changes underlying GlyR channel gating. The functional state assignments were validated by molecular dynamics simulations of the structures incorporated in a lipid bilayer. Observed permeation events are in agreement with the anion selectivity of the channel and the reported single-channel conductance of GlyR. These studies establish the structural basis for gating, selectivity, and single-channel conductance of GlyR in a physiological environment.

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

The Journal of General Physiology ◽

10.1085/jgp.201511478 ◽

2015 ◽

Vol 146 (4) ◽

pp. 323-340 ◽

Cited By ~ 11

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.

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Structural studies on leukotriene A4 hydrolases reveal their catalytic mechanism

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095862 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C413-C413

Author(s):

Mahmudul Hasan ◽

Agnes Rinaldo-Matthis ◽

Marjolein Thunnissen

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Active Site ◽

Epoxide Hydrolase ◽

Aminopeptidase Activity ◽

Molecular Architecture ◽

Inhibitor Binding ◽

Induced Fit ◽

Human Enzyme ◽

Leukotriene A4

Vertebrate leukotriene A4 hydrolases are zinc metalloenzymes with an epoxide hydrolase and aminopeptidase activity belonging to the M1 family of aminopeptidases. Bestatin, an amino peptidase inhibitor, can inhibit both the activities. The human enzyme produces LTB4, a powerful mediator of inflammation and is implicated in a wide variety of rheumatoid diseases. The yeast homolog scLTA4H contains only a rudimentary epoxide hydrolase activity. Both the structure of the human enzyme and recently the structure of scLTA4H and have been solved to investigate the molecular architecture of the active site both with and without inhibitor Bestatin. The structure of scLTA4H shows large domain movements creating an open active site. In the human enzyme the LTA4 binding side is a narrow hydrophobic channel. Upon inhibitor a domain shifts occurs and the final binding pocket is formed. The fact that scLTA4H displays this induced fit is an interesting observation. Many members of the M1 family seem to display a certain degree of induced fit, a feature, which however, has never been observed for humLTA4H. Our recent solution SAXS studies show that humLTA4H does not make any conformational changes upon inhibitor binding which is consistent with our previous speculation that it functions by a lock and key mechanism rather than induced fit and is better suited to supply the protective and precise environment for hydrolysis of LTA4 into LTB4. On the other hand Xenopus LTA4H shows conformational change in the higher/wide angular region ( >1 nm-1) and decrease in Porod volume of approximately 20 nm3 but no change in Rg or Dmax was observed. It is also observed that like in crystal structure Xenopus LTA4H forms dimer in solution. Similarly scLTA4H forms dimer in solution, which is unlike the crystal structure, and also make conformational changes upon inhibitor binding. Taken together, Xenopus and scLTA4H makes more compact form, with decrease in flexibility, to perform it's catalytic action.

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Force-Generating Mechanism of Axonemal Dynein in Solo and Ensemble

International Journal of Molecular Sciences ◽

10.3390/ijms21082843 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2843

Author(s):

Kenta Ishibashi ◽

Hitoshi Sakakibara ◽

Kazuhiro Oiwa

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Current Knowledge ◽

Expression System ◽

Molecular Architecture ◽

Electron Microscopic ◽

Axonemal Dynein ◽

Cilia And Flagella ◽

Amount Of Knowledge

In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowledge and perspectives of the force-generating mechanism of axonemal dyneins in solo and in ensemble.

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Molecular architecture of black widow spider neurotoxins

Nature Communications ◽

10.1038/s41467-021-26562-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Minghao Chen ◽

Daniel Blum ◽

Lena Engelhard ◽

Stefan Raunser ◽

Richard Wagner ◽

...

Keyword(s):

Conformational Changes ◽

Calcium Flux ◽

Membrane Insertion ◽

Molecular Architecture ◽

Cyclic Mechanism ◽

High Resolution Structure ◽

Specific Receptors ◽

Β Sheet ◽

Widow Spider ◽

Resolution Structure

AbstractLatrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

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Structures of the fungal dynamin-related protein Vps1 reveal a unique, open helical architecture

The Journal of Cell Biology ◽

10.1083/jcb.201712021 ◽

2018 ◽

Vol 217 (10) ◽

pp. 3608-3624 ◽

Cited By ~ 4

Author(s):

Natalia V. Varlakhanova ◽

Frances J.D. Alvarez ◽

Tyler M. Brady ◽

Bryan A. Tornabene ◽

Christopher J. Hosford ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Self Assembly ◽

Related Protein ◽

Membrane Remodeling ◽

Helical Structures ◽

Gtp Hydrolysis ◽

Cellular Targets ◽

Related Proteins

Dynamin-related proteins (DRPs) are large multidomain GTPases required for diverse membrane-remodeling events. DRPs self-assemble into helical structures, but how these structures are tailored to their cellular targets remains unclear. We demonstrate that the fungal DRP Vps1 primarily localizes to and functions at the endosomal compartment. We present crystal structures of a Vps1 GTPase–bundle signaling element (BSE) fusion in different nucleotide states to capture GTP hydrolysis intermediates and concomitant conformational changes. Using cryoEM, we determined the structure of full-length GMPPCP-bound Vps1. The Vps1 helix is more open and flexible than that of dynamin. This is due to further opening of the BSEs away from the GTPase domains. A novel interface between adjacent GTPase domains forms in Vps1 instead of the contacts between the BSE and adjacent stalks and GTPase domains as seen in dynamin. Disruption of this interface abolishes Vps1 function in vivo. Hence, Vps1 exhibits a unique helical architecture, highlighting structural flexibilities of DRP self-assembly.

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Molecular architecture of black widow spider neurotoxins

10.1101/2021.04.19.440504 ◽

2021 ◽

Author(s):

Minghao Chen ◽

Daniel Blum ◽

Lena Engelhard ◽

Stefan Raunser ◽

Richard Wagner ◽

...

Keyword(s):

Conformational Changes ◽

Calcium Flux ◽

Membrane Insertion ◽

Molecular Architecture ◽

Cyclic Mechanism ◽

High Resolution Structure ◽

Specific Receptors ◽

Β Sheet ◽

Widow Spider ◽

Resolution Structure

AbstractLatrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ɛ-latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear.Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

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Molecular architecture of 40S initiation complexes on the Hepatitis C virus IRES: from ribosomal attachment to eIF5B-mediated reorientation of initiator tRNA

10.1101/2021.12.10.472117 ◽

2021 ◽

Author(s):

Zuben P. Brown ◽

Irina S. Abaeva ◽

Swastik De ◽

Christopher U.T. Hellen ◽

Tatyana V. Pestova ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Conformational Changes ◽

Dynamic Network ◽

Molecular Architecture ◽

Entry Site ◽

Internal Ribosome Entry ◽

Initiation Factors ◽

Direct Binding ◽

Subunit Joining

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Direct binding of the IRES to the 40S subunit places the initiation codon into the P site, where it base-pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Then, eIF5 and eIF5B mediate subunit joining. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. Here, we present cryo-EM structures of IRES initiation complexes at resolutions up to 3.5 Å that cover all major stages from initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role for IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes that prepare them for subunit joining.

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