scholarly journals Motility and microtubule depolymerization mechanisms of the Kinesin-8 motor, KIF19A

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Doudou Wang ◽  
Ryo Nitta ◽  
Manatsu Morikawa ◽  
Hiroaki Yajima ◽  
Shigeyuki Inoue ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Dual Function ◽  
Microtubule Depolymerization ◽  
Multiple Strategies ◽  
Cryo Electron Microscopy ◽  
Cilium Length ◽  
Dual Functions

The kinesin-8 motor, KIF19A, accumulates at cilia tips and controls cilium length. Defective KIF19A leads to hydrocephalus and female infertility because of abnormally elongated cilia. Uniquely among kinesins, KIF19A possesses the dual functions of motility along ciliary microtubules and depolymerization of microtubules. To elucidate the molecular mechanisms of these functions we solved the crystal structure of its motor domain and determined its cryo-electron microscopy structure complexed with a microtubule. The features of KIF19A that enable its dual function are clustered on its microtubule-binding side. Unexpectedly, a destabilized switch II coordinates with a destabilized L8 to enable KIF19A to adjust to both straight and curved microtubule protofilaments. The basic clusters of L2 and L12 tether the microtubule. The long L2 with a characteristic acidic-hydrophobic-basic sequence effectively stabilizes the curved conformation of microtubule ends. Hence, KIF19A utilizes multiple strategies to accomplish the dual functions of motility and microtubule depolymerization by ATP hydrolysis.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

scholarly journals Cryo–electron microscopy structure of the antidiuretic hormone arginine-vasopressin V2 receptor signaling complex

2021 ◽  
Vol 7 (21) ◽  
pp. eabg5628
Author(s):  
Julien Bous ◽  
Hélène Orcel ◽  
Nicolas Floquet ◽  
Cédric Leyrat ◽  
Joséphine Lai-Kee-Him ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Antidiuretic Hormone ◽  
Arginine Vasopressin ◽  
Molecular Mechanisms ◽  
Particle Analysis ◽  
Resonance Spectroscopy ◽  
Gpcr Signaling ◽  
Signaling Complex ◽  
Cryo Electron Microscopy ◽  
V2 Receptor

The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein–coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo–electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.

scholarly journals Positive and negative intra-molecular modulation in a dual-cassette RNA helicase

2019 ◽  
Author(s):  
Karen Vester ◽  
Karine F. Santos ◽  
Benno Kuropka ◽  
Christoph Weise ◽  
Markus C. Wahl
Keyword(s):  
Crystal Structure ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Rna Helicase ◽  
Negative Influence ◽  
Disulfide Bridges ◽  
Relative Positioning ◽  
Distinct Subgroup ◽  
Terminal Unit ◽  
Molecular Modulation

ABSTRACTRNA helicase Brr2 is required for the activation of the spliceosome prior to the first catalytic step of splicing. Brr2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the N-terminal cassette of Brr2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudo-enzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms, by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive. Here, we show that N- and C-terminal cassettes adopt vastly different relative orientations in a crystal structure of Brr2 in complex with an activating domain of the spliceosomal Prp8 protein as compared to the crystal structure of isolated Brr2. Likewise, the cassettes occupy different relative positions and engage in different inter-cassette contacts during different stages of splicing. Engineered disulfide bridges that lock the cassettes in two different relative orientations have opposite effects on RNA-related activities of the N-terminal cassette compared to the unrestrained protein. Moreover, different relative positioning of the cassettes strongly influences ATP hydrolysis by the N-terminal cassette. Our results demonstrate that the inactive C-terminal cassette of Brr2 can exert both positive and negative influence on the active N-terminal helicase unit from a distance.

scholarly journals The Primary Enveloped Virion of Herpes Simplex Virus 1: Its Role in Nuclear Egress

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
William W. Newcomb ◽  
Juan Fontana ◽  
Dennis C. Winkler ◽  
Naiqian Cheng ◽  
J. Bernard Heymann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Membrane ◽  
Molecular Mechanisms ◽  
Weak Interactions ◽  
Nuclear Pore ◽  
Nuclear Egress ◽  
Cryo Electron Microscopy ◽  
Perinuclear Space ◽  
Infected Cells ◽  
Hsv 1

ABSTRACTMany viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. We investigated the associated molecular mechanisms by isolating PEVs from US3-null-infected cells and imaging them by cryo-electron microscopy and tomography. (pUS3 is a viral protein kinase in whose absence PEVs accumulate in the perinuclear space.) Unlike mature extracellular virions, PEVs have very few glycoprotein spikes. PEVs are ~20% smaller than mature virions, and the little space available between the capsid and the NEC layer suggests that most tegument proteins are acquired later in the egress pathway. Previous studies have proposed that NEC is organized as hexamers in honeycomb arrays in PEVs, but we find arrays of heptameric rings in extracts from US3-null-infected cells. In a PEV, NEC contacts the capsid predominantly via the pUL17/pUL25 complexes which are located close to the capsid vertices. Finally, the NEC layer dissociates from the capsid as it leaves the nucleus, possibly in response to pUS3-mediated phosphorylation. Overall, nuclear egress emerges as a process driven by a program of multiple weak interactions.IMPORTANCEOn its maturation pathway, the newly formed HSV-1 nucleocapsid must traverse the nuclear envelope, while respecting the integrity of that barrier. Nucleocapsids (125 nm in diameter) are too large to pass through the nuclear pore complexes that conduct most nucleocytoplasmic traffic. It is now widely accepted that the process involves envelopment/de-envelopment of a key intermediate—the primary enveloped virion. In wild-type infections, PEVs are short-lived, which has impeded study. Using a mutant that accumulates PEVs in the perinuclear space, we were able to isolate PEVs in sufficient quantity for structural analysis by cryo-electron microscopy and tomography. The findings not only elucidate the maturation pathway of an important human pathogen but also have implications for cellular processes that involve the trafficking of large macromolecular complexes.

scholarly journals Cryo-EM Reveals the Structural Basis of Microtubule Depolymerization by Kinesin-13s

2017 ◽  
Author(s):  
Matthieu P. M. H. Benoit ◽  
Ana B. Asenjo ◽  
Hernando Sosa
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Distinct Group ◽  
Atomic Resolution ◽  
Structural Basis ◽  
Structural Elements ◽  
Microtubule Depolymerization ◽  
Cryo Electron Microscopy ◽  
Kinesin Superfamily ◽  
Motile Activity

SummaryKinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promotes microtubule depolymerization and lacks motile activity. The molecular mechanism by which the kinesins depolymerize microtubules and are adapted to perform a seemingly very different activity from other kinesins is still unclear. To address this issue we obtained near atomic resolution cryo-electron microscopy (cryo-EM) structures of Drosophila melanogaster kinesin-13 KLP10A constructs bound to curved or straight tubulin in different nucleotide states. The structures show how nucleotide induced conformational changes near the catalytic site are coupled with kinesin-13-specific structural elements to induce tubulin curvature leading to microtubule depolymerization. The data highlight a modular structure that allows similar kinesin core motor-domains to be used for different functions, such as motility or microtubule depolymerization.

scholarly journals Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alexandria N Miller ◽  
George Vaisey ◽  
Stephen B Long
Keyword(s):  
Electron Microscopy ◽  
Ion Channels ◽  
Molecular Mechanisms ◽  
Peptide Binding ◽  
Retinal Disease ◽  
Structural Similarity ◽  
Cryo Electron Microscopy ◽  
Cl Channels ◽  
Pore Lining ◽  
Protein Shell

Bestrophin (BEST1-4) ligand-gated chloride (Cl-) channels are activated by calcium (Ca2+). Mutation of BEST1 causes retinal disease. Partly because bestrophin channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the channel’s principal gating states. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a neck of the pore through a concertina of amino acid rearrangements. The neck serves as both the activation and the inactivation gate. Ca2+ binding instigates opening of the neck through allosteric means whereas inactivation peptide binding induces closing. An aperture within the otherwise wide pore controls anion permeability. The studies define a new molecular paradigm for gating among ligand-gated ion channels.

scholarly journals Cryo-Electron Microscopy of Arabidopsis thaliana Phytochrome A in Its Pr State Reveals Head-to-Head Homodimeric Architecture

2021 ◽  
Vol 12 ◽  
Author(s):  
Weixiao Yuan Wahlgren ◽  
David Golonka ◽  
Sebastian Westenhoff ◽  
Andreas Möglich
Keyword(s):  
Electron Microscopy ◽  
Arabidopsis Thaliana ◽  
Electron Density ◽  
Molecular Mechanisms ◽  
Signal Propagation ◽  
Full Length ◽  
Particle Analysis ◽  
Phytochrome A ◽  
Cryo Electron Microscopy ◽  
Bacterial Phytochromes

Phytochrome photoreceptors regulate vital adaptations of plant development, growth, and physiology depending on the ratio of red and far-red light. The light-triggered Z/E isomerization of a covalently bound bilin chromophore underlies phytochrome photoconversion between the red-absorbing Pr and far-red-absorbing Pfr states. Compared to bacterial phytochromes, the molecular mechanisms of signal propagation to the C-terminal module and its regulation are little understood in plant phytochromes, not least owing to a dearth of structural information. To address this deficit, we studied the Arabidopsis thaliana phytochrome A (AtphyA) at full length by cryo-electron microscopy (cryo-EM). Following heterologous expression in Escherichia coli, we optimized the solvent conditions to overcome protein aggregation and thus obtained photochemically active, near-homogenous AtphyA. We prepared grids for cryo-EM analysis of AtphyA in its Pr state and conducted single-particle analysis. The resulting two-dimensional class averages and the three-dimensional electron density map at 17 Å showed a homodimeric head-to-head assembly of AtphyA. Docking of domain structures into the electron density revealed a separation of the AtphyA homodimer at the junction of its photosensor and effector modules, as reflected in a large void in the middle of map. The overall architecture of AtphyA resembled that of bacterial phytochromes, thus hinting at commonalities in signal transduction and mechanism between these receptors. Our work paves the way toward future studies of the structure, light response, and interactions of full-length phytochromes by cryo-EM.

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.

Application of cryo-electron microscopy for investigation of Bax-induced pores in apoptosis

2017 ◽  
Vol 6 (1) ◽  
pp. 47-55
Author(s):  
Tomomi Kuwana
Keyword(s):  
Electron Microscopy ◽  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Experimental System ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Cryo Electron Microscopy

AbstractMitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis, the molecular mechanisms of which have been a subject of intensive study. This process is important for therapeutic intervention in various diseases, such as cancer. Pro-apoptotic Bax and Bak are functionally redundant and structurally homologous. When activated at the mitochondrial outer membrane, they cause the membrane to permeabilize and release apoptogenic proteins from the intermembrane space. To unravel the molecular mechanisms of this unique and important event, we systematically reduced the experimental system. Simple outer membrane vesicles and liposomes recapitulated many features of MOMP. Although conventional transmission electron microscopy could not detect any membrane changes during MOMP in these vesicles, cryo-electron microscopy successfully revealed Bax-induced delicate pores, owing to its ability to preserve native, hydrated membrane structure. The data are consistent with the idea that Bax is unfolded and embedded in the bilayer and deforms the membrane to form a large pore. Together with the biochemical and structure data in the literature, we now have more comprehensive models of the key function of Bax. We hope that new tools, such as lipid nanodiscs, will give us an atomic-level resolution and finally solve Bax structure in the membrane, where it functions.

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