The ryanodine receptor provides high throughput Ca2+-release but is precisely regulated by networks of associated proteins: a focus on proteins relevant to phosphorylation

2015 ◽  
Vol 43 (3) ◽  
pp. 426-433 ◽  
Author(s):  
Fiona O'Brien ◽  
Elisa Venturi ◽  
Rebecca Sitsapesan
Keyword(s):  
Spatial Organization ◽  
Ryanodine Receptors ◽  
Integrated System ◽  
Fine Tuning ◽  
Control Mechanisms ◽  
Fk 506 ◽  
High Resolution Structure ◽  
Associated Proteins ◽  
And Function ◽  
Resolution Structure

Once opened, ryanodine receptors (RyR) are efficient pathways for the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR). The precise nature of the Ca2+-release event, however, requires fine-tuning for the specific process and type of cell involved. For example, the spatial organization of RyRs, the luminal [Ca2+] and the influence of soluble regulators that fluctuate under physiological and pathophysiological control mechanisms, all affect the amplitude and duration of RyR Ca2+ fluxes. Various proteins are docked tightly to the huge bulky structure of RyR and there is growing evidence that, together, they provide a sophisticated and integrated system for regulating RyR channel gating. This review focuses on those proteins that are relevant to phosphorylation of RyR channels with particular reference to the cardiac isoform of RyR (RyR2). How phosphorylation of RyR affects channel activity and whether proteins such as the FK-506 binding proteins (FKBP12 and FKBP12.6) are involved, have been highly controversial subjects for more than a decade. But that is expected given the large number of participating proteins, the relevance of phosphorylation in heart failure and inherited arrhythmic diseases, and the frustrations of predicting relationships between structure and function before the advent of a high resolution structure of RyR.

Towards understanding the catalytic core structure of the spliceosome

2005 ◽  
Vol 33 (3) ◽  
pp. 447-449 ◽  
Author(s):  
S.E. Butcher ◽  
D.A. Brow
Keyword(s):  
Structure And Function ◽  
Mrna Splicing ◽  
Catalytic Core ◽  
Coding Regions ◽  
High Resolution Structure ◽  
Associated Proteins ◽  
And Function ◽  
Mrna Precursor ◽  
Precursor Mrna ◽  
Resolution Structure

The spliceosome catalyses the splicing of nuclear pre-mRNA (precursor mRNA) in eukaryotes. Pre-mRNA splicing is essential to remove internal non-coding regions of pre-mRNA (introns) and to join the remaining segments (exons) into mRNA before translation. The spliceosome is a complex assembly of five RNAs (U1, U2, U4, U5 and U6) and many dozens of associated proteins. Although a high-resolution structure of the spliceosome is not yet available, inroads have been made towards understanding its structure and function. There is growing evidence suggesting that U2 and U6 RNAs, of the five, may contribute to the catalysis of pre-mRNA splicing. In this review, recent progress towards understanding the structure and function of U2 and U6 RNAs is summarized.

scholarly journals Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge

2019 ◽  
Author(s):  
Ariel Ben-Bassat ◽  
Moshe Giladi ◽  
Yoni Haitin
Keyword(s):  
Cyclic Nucleotide ◽  
Spatial Organization ◽  
Family Members ◽  
Salt Bridge ◽  
Nucleotide Binding ◽  
Spectroscopic Studies ◽  
Structural Mechanism ◽  
Cyclic Nucleotide Binding ◽  
High Resolution Structure ◽  
Resolution Structure

AbstractHuman KCNH2 (hKCNH2, Ether-à-go-go (EAG)-Related Gene, hERG) are best known for their role in cardiac action potentials repolarization and have key roles in various pathologies. As other KCNH family members, hKCNH2 contains a unique intracellular complex crucial for channel function, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD). Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif (ILM), in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channels regulation by cyclic nucleotides. While significant advancements in structural and functional characterizations of the CNBHD of KCNH channels have been made, a high-resolution structure of the hKCNH2 intracellular complex was missing. Here, we report the 1.5 Å resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the ILM is preserved within the β-roll region. Moreover, measurements of small-angle X-ray scattering profile in solution, as well as comparison with a recent nuclear magnetic resonance (NMR) analysis of hKCNH2, revealed high agreement with the structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863), which was not previously resolved in the NMR and cryogenic electron microscopy (cryo-EM) structures. Strikingly, electrophysiological analysis of charge reversal mutations revealed its crucial role for hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance. Together with the available structure of the mouse KCNH1 intracellular complex, and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the ILM and the maintenance of the intracellular complex interface.SummaryHuman KCNH2 are key channels governing cardiac repolarization. Here, a 1.5 Å resolution structure of their cyclic nucleotide-binding homology domain is presented. Structural analysis and electrophysiological validation reveal a novel salt-bridge, playing an important role in hKCNH2 functional regulation.

scholarly journals Microtubule Regulation and Function during Virus Infection

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Mojgan H. Naghavi ◽  
Derek Walsh
Keyword(s):  
Posttranslational Modifications ◽  
Intracellular Transport ◽  
Spatial Organization ◽  
Virus Particles ◽  
Cellular Processes ◽  
Wide Range ◽  
Associated Proteins ◽  
And Function ◽  
Induced Changes ◽  
Growth Pause

ABSTRACT Microtubules (MTs) form a rapidly adaptable network of filaments that radiate throughout the cell. These dynamic arrays facilitate a wide range of cellular processes, including the capture, transport, and spatial organization of cargos and organelles, as well as changes in cell shape, polarity, and motility. Nucleating from MT-organizing centers, including but by no means limited to the centrosome, MTs undergo rapid transitions through phases of growth, pause, and catastrophe, continuously exploring and adapting to the intracellular environment. Subsets of MTs can become stabilized in response to environmental cues, acquiring distinguishing posttranslational modifications and performing discrete functions as specialized tracks for cargo trafficking. The dynamic behavior and organization of the MT array is regulated by MT-associated proteins (MAPs), which include a subset of highly specialized plus-end-tracking proteins (+TIPs) that respond to signaling cues to alter MT behavior. As pathogenic cargos, viruses require MTs to transport to and from their intracellular sites of replication. While interactions with and functions for MT motor proteins are well characterized and extensively reviewed for many viruses, this review focuses on MT filaments themselves. Changes in the spatial organization and dynamics of the MT array, mediated by virus- or host-induced changes to MT regulatory proteins, not only play a central role in the intracellular transport of virus particles but also regulate a wider range of processes critical to the outcome of infection.

scholarly journals Structure and function of the bacterial protein toxin phenomycin

2019 ◽  
Author(s):  
Bente K. Hansen ◽  
Camilla K. Larsen ◽  
Jacob T. Nielsen ◽  
Esben B. Svenningsen ◽  
Lan B. Van ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Endosomal Escape ◽  
Bacterial Protein ◽  
Direct Inhibition ◽  
Uptake Mechanism ◽  
Protein Toxin ◽  
Limiting Step ◽  
High Resolution Structure ◽  
And Function ◽  
Resolution Structure

SummaryPhenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime towards mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phenomycin have however remained unanswered. In this paper, we have used morphological profiling to show that direct inhibition of translation underlies the toxicity of phenomycin in cells. We have performed studies of the cellular uptake mechanism of phenomycin, showing that endosomal escape is the toxicity-limiting step, and we have solved a solution phase high-resolution structure of the protein using NMR spectroscopy. Through bioinformatic as well as functional comparisons between phenomycin and two homologs, we have identified a peptide segment, which constitutes one of two loops in the structure, that is critical for the toxicity of phenomycin.

scholarly journals GeRelion: GPU-enhanced parallel implementation of single particle cryo-EM image processing

2016 ◽  
Author(s):  
Huayou Su ◽  
Wen Wen ◽  
Xiaoli Du ◽  
Xicheng Lu ◽  
Maofu Liao ◽  
...  
Keyword(s):  
Image Processing ◽  
Single Particle ◽  
Parallel Implementation ◽  
Data Sets ◽  
Structural Details ◽  
High Resolution Structure ◽  
Molecular Machinery ◽  
Speed Up ◽  
And Function ◽  
Resolution Structure

Single particle cryo-EM emerges as a powerful and versatile method to characterize the structure and function of macromolecules, revealing the structural details of critical molecular machinery inside the cells. RELION is a widely used EM image processing software, and most of the recently published single particle cryo-EM structures were generated by using RELION. Due to the massive computational loads and the growing demands for processing much larger cryo-EM data sets, there is a pressing need to speed up image processing. Here we present GeRelion (https://github.com/gpu-pdl¬nudt/GeRelion), an efficient parallel implementation of RELION on GPU system. In the performance tests using two cryo-EM data sets, GeRelion on 4 or 8 GPU cards outperformed RELION on 256 CPU cores, demonstrating dramatically improved speed and superb scalability. By greatly accelerating single particle cryo-EM structural analysis, GeRelion will facilitate both high resolution structure determination and dissection of mixed conformations of dynamic molecular machines.

scholarly journals Cryo-EM structure of Mycobacterium smegmatis DyP-loaded encapsulin

2021 ◽  
Vol 118 (16) ◽  
pp. e2025658118
Author(s):  
Yanting Tang ◽  
An Mu ◽  
Yuying Zhang ◽  
Shan Zhou ◽  
Weiwei Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mycobacterium Smegmatis ◽  
Structural Features ◽  
Antituberculosis Drug ◽  
Potential Mechanism ◽  
Cargo Protein ◽  
High Resolution Structure ◽  
Axis Of Symmetry ◽  
And Function ◽  
Resolution Structure

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.

scholarly journals Topography of Lipid Droplet-Associated Proteins: Insights from Freeze-Fracture Replica Immunogold Labeling

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Horst Robenek ◽  
Insa Buers ◽  
Mirko J. Robenek ◽  
Oliver Hofnagel ◽  
Anneke Ruebel ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
Lipid Droplets ◽  
Spatial Organization ◽  
Freeze Fracture ◽  
Protein Composition ◽  
Immunogold Labeling ◽  
Intracellular Lipids ◽  
Associated Proteins ◽  
Developing Area ◽  
And Function

Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how freeze-fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with freeze-fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies.

scholarly journals Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design

2020 ◽  
Author(s):  
Dénes Berta ◽  
Magd Badaoui ◽  
Pedro J. Buigues ◽  
Sam Alexander Martino ◽  
Andrei V. Pisliakov ◽  
...  
Keyword(s):  
Drug Target ◽  
Md Simulation ◽  
Structural Information ◽  
Rna Helicase ◽  
Atomic Level ◽  
Virus Rna ◽  
High Resolution Structure ◽  
And Function ◽  
Structural Insights ◽  
Resolution Structure

ABSTRACTHaving claimed over 1 million lives worldwide to date, the ongoing COVID-19 pandemic has created one of the biggest challenges to develop an effective drug to treat infected patients. Among all the proteins expressed by the virus, RNA helicase is a fundamental protein for viral replication, and it is highly conserved among the coronaviridae family. To date, there is no high-resolution structure of helicase bound with ATP and RNA. We present here structural insights and molecular dynamics (MD) simulation results of the SARS-CoV-2 RNA helicase both in its apo form and in complex with its natural substrates. Our structural information of the catalytically competent helicase complex provides valuable insights for the mechanism and function of this enzyme at the atomic level, a key to develop specific inhibitors for this potential COVID-19 drug target.

scholarly journals Role of cytochrome c oxidase nuclear-encoded subunits in health and disease

2020 ◽  
pp. 947-965
Author(s):  
K Čunátová ◽  
D Pajuelo Reguera ◽  
J Houštěk ◽  
T Mráček ◽  
P Pecina
Keyword(s):  
Electron Transport ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Fine Tuning ◽  
Transport Chain ◽  
Associated Proteins ◽  
And Function ◽  
Structural Levels

Cytochrome c oxidase (COX), the terminal enzyme of mitochondrial electron transport chain, couples electron transport to oxygen with generation of proton gradient indispensable for the production of vast majority of ATP molecules in mammalian cells. The review summarizes current knowledge of COX structure and function of nuclear-encoded COX subunits, which may modulate enzyme activity according to various conditions. Moreover, some nuclear-encoded subunits posess tissue-specific and development-specific isoforms, possibly enabling fine-tuning of COX function in individual tissues. The importance of nuclear-encoded subunits is emphasized by recently discovered pathogenic mutations in patients with severe mitopathies. In addition, proteins substoichiometrically associated with COX were found to contribute to COX activity regulation and stabilization of the respiratory supercomplexes. Based on the summarized data, a model of three levels of quaternary COX structure is postulated. Individual structural levels correspond to subunits of the i) catalytic center, ii) nuclear-encoded stoichiometric subunits and iii) associated proteins, which may constitute several forms of COX with varying composition and differentially regulated function.

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