scholarly journals Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail

2020 ◽  
Vol 6 (33) ◽  
pp. eabb0147 ◽  
Author(s):  
Yuxia Zhang ◽  
Michio Inoue ◽  
Akihisa Tsutsumi ◽  
Satoshi Watanabe ◽  
Tomohiro Nishizawa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Calcium Homeostasis ◽  
Critical Role ◽  
Kinetic Properties ◽  
Cryo Electron Microscopy ◽  
Domain Arrangement ◽  
Tm Domain ◽  
Conformational Regulation ◽  
Multiple Conformations

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo–electron microscopy (cryo-EM) structures of human SERCA2b in E1∙2Ca2+–adenylyl methylenediphosphonate (AMPPCP) and E2-BeF3− states at 2.9- and 2.8-Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7 and approaches the luminal loop flanked by TM7 and TM8. While the LE served to stabilize the cytosolic and TM domain arrangement of SERCA2b, deletion of the LE rendered the overall conformation resemble that of SERCA1a and SERCA2a and allowed multiple conformations. Thus, the LE appears to play a critical role in conformational regulation in SERCA2b, which likely explains the different kinetic properties of SERCA2b from those of other isoforms lacking the LE.

scholarly journals Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail

2020 ◽  
Author(s):  
Yuxia Zhang ◽  
Michio Inoue ◽  
Akihisa Tsutsumi ◽  
Satoshi Watanabe ◽  
Tomohiro Nishizawa ◽  
...  
Keyword(s):  
Calcium Homeostasis ◽  
Conformational Transition ◽  
Kinetic Properties ◽  
Cellular Calcium ◽  
Domain Arrangement ◽  
Tm Domain ◽  
Multiple Conformations

AbstractSERCA2b is a Ca2+-ATPase that pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo-EM structures of human SERCA2b in E1·2Ca2+-AMPPCP and E2-BeF3- states at 2.9 and 2.8 Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7, and approaches the luminal loop flanked by TM7 and TM8. Upon deletion of the LE, the cytosolic- and TM-domain arrangement of SERCA2b resembled that of SERCA1a, resulting in multiple conformations. The LE regulates the conformational transition between the E1·2Ca2+-ATP and E2P states, explaining the different kinetic properties of SERCA2b from other isoforms lacking the LE.One sentence summaryCryo-EM structures of SERCA2b at 2.8-2.9 Å resolutions reveal how the luminal extension tail regulates its activity.

scholarly journals Structure of BipA in GTP form bound to the ratcheted ribosome

2015 ◽  
Vol 112 (35) ◽  
pp. 10944-10949 ◽  
Author(s):  
Veerendra Kumar ◽  
Yun Chen ◽  
Rya Ero ◽  
Tofayel Ahmed ◽  
Jackie Tan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Environmental Changes ◽  
Critical Role ◽  
Protein A ◽  
Cellular Responses ◽  
Cryo Electron Microscopy ◽  
The Family ◽  
Domain Arrangement ◽  
Inducible Protein ◽  
A Site

BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3′,5′-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation.

scholarly journals Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.

2021 ◽  
Author(s):  
Philipp Milkereit ◽  
Gisela Pöll ◽  
Michael Pilsl ◽  
Joachim Griesenbeck ◽  
Herbert Tschochner
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Protein Assembly ◽  
Functional Coupling ◽  
Large Ribosomal Subunit ◽  
Cryo Electron Microscopy ◽  
Intrinsic Quality ◽  
Domain Arrangement ◽  
The Stability

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

scholarly journals Structure of the posttranslational Sec protein-translocation channel complex from yeast

Science ◽  
2018 ◽  
Vol 363 (6422) ◽  
pp. 84-87 ◽  
Author(s):  
Samuel Itskanov ◽  
Eunyong Park
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Binding Sites ◽  
Protein Translocation ◽  
Secretory Proteins ◽  
Conducting Channel ◽  
Cryo Electron Microscopy ◽  
Lateral Gate

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

scholarly journals Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0252497
Author(s):  
Gisela Pöll ◽  
Michael Pilsl ◽  
Joachim Griesenbeck ◽  
Herbert Tschochner ◽  
Philipp Milkereit
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Protein Assembly ◽  
Functional Coupling ◽  
Large Ribosomal Subunit ◽  
Cryo Electron Microscopy ◽  
Intrinsic Quality ◽  
Domain Arrangement ◽  
The Stability

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

scholarly journals Cryo–electron microscopy structures of human oligosaccharyltransferase complexes OST-A and OST-B

Science ◽  
2019 ◽  
Vol 366 (6471) ◽  
pp. 1372-1375 ◽  
Author(s):  
Ana S. Ramírez ◽  
Julia Kowal ◽  
Kaspar P. Locher
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Secretory Proteins ◽  
Catalytic Subunits ◽  
Protein Substrates ◽  
Cryo Electron Microscopy ◽  
Structural Differences ◽  
High Mannose ◽  
Equivalent Region ◽  
High Mannose Glycan

Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum. Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner. Here, we present high-resolution cryo–electron microscopy structures of human OST-A and OST-B. Although they have similar overall architectures, structural differences in the catalytic subunits STT3A and STT3B facilitate contacts to distinct OST subunits, DC2 in OST-A and MAGT1 in OST-B. In OST-A, interactions with TMEM258 and STT3A allow ribophorin-I to form a four-helix bundle that can bind to a translating ribosome, whereas the equivalent region is disordered in OST-B. We observed an acceptor peptide and dolichylphosphate bound to STT3B, but only dolichylphosphate in STT3A, suggesting distinct affinities of the two OST complexes for protein substrates.

scholarly journals Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies

2020 ◽  
Vol 6 (29) ◽  
pp. eaba8105 ◽  
Author(s):  
Kanae Demura ◽  
Tsukasa Kusakizako ◽  
Wataru Shihoya ◽  
Masahiro Hiraizumi ◽  
Kengo Nomura ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Caenorhabditis Elegans ◽  
Adenosine Triphosphate ◽  
Calcium Homeostasis ◽  
Transmembrane Domain ◽  
Atp Release ◽  
Channel Pore ◽  
Cryo Electron Microscopy ◽  
Chimeric Construct ◽  
Intersubunit Interactions

Calcium homeostasis modulator (CALHM) family proteins are Ca2+-regulated adenosine triphosphate (ATP)–release channels involved in neural functions including neurotransmission in gustation. Here, we present the cryo–electron microscopy (EM) structures of killifish CALHM1, human CALHM2, and Caenorhabditis elegans CLHM-1 at resolutions of 2.66, 3.4, and 3.6 Å, respectively. The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction site at the channel pore in the open state and modulates the ATP conductance. The CALHM2 undecamer and CLHM-1 nonamer structures show the different oligomeric stoichiometries among CALHM homologs. We further report the cryo-EM structures of the chimeric construct, revealing that the intersubunit interactions at the transmembrane domain (TMD) and the TMD–intracellular domain linker define the oligomeric stoichiometry. These findings advance our understanding of the ATP conduction and oligomerization mechanisms of CALHM channels.

scholarly journals Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex

2021 ◽  
Author(s):  
Brandon Malone ◽  
James Chen ◽  
Qi Wang ◽  
Eliza Llewellyn ◽  
Young Joo Choi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Rna Polymerase ◽  
Critical Role ◽  
Structural Basis ◽  
Rna Dependent Rna Polymerase ◽  
Present Evidence ◽  
Cryo Electron Microscopy ◽  
Dynamics Simulations ◽  
Transcription And Replication

AbstractBacktracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein crosslinking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3’-segment of the product-RNA generated by backtracking extrudes through the RdRp NTP-entry tunnel, that a mismatched nucleotide at the product-RNA 3’-end frays and enters the NTP-entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.Significance StatementThe COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome is replicated and transcribed by its RNA-dependent RNA polymerase (RdRp), which is the target for antivirals such as remdesivir. We use a combination of approaches to show that backtracking (backwards motion of the RdRp on the template RNA) is a feature of SARS-CoV-2 replication/transcription. Backtracking may play a critical role in proofreading, a crucial process for SARS-CoV-2 resistance against many antivirals.

scholarly journals Molecular principles of assembly, activation, and inhibition in epithelial sodium channel

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Sigrid Noreng ◽  
Richard Posert ◽  
Arpita Bharadwaj ◽  
Alexandra Houser ◽  
Isabelle Baconguis
Keyword(s):  
Functional Module ◽  
Critical Role ◽  
Epithelial Sodium Channels ◽  
Inhibitory Peptide ◽  
Α Subunit ◽  
Cryo Electron Microscopy ◽  
Domain Arrangement ◽  
Molecular Bases ◽  
Regulatory Sites

The molecular bases of heteromeric assembly and link between Na+ self-inhibition and protease-sensitivity in epithelial sodium channels (ENaCs) are not fully understood. Previously, we demonstrated that ENaC subunits – α, β, and γ – assemble in a counterclockwise configuration when viewed from outside the cell with the protease-sensitive GRIP domains in the periphery (Noreng et al., 2018). Here we describe the structure of ENaC resolved by cryo-electron microscopy at 3 Å. We find that a combination of precise domain arrangement and complementary hydrogen bonding network defines the subunit arrangement. Furthermore, we determined that the α subunit has a primary functional module consisting of the finger and GRIP domains. The module is bifurcated by the α2 helix dividing two distinct regulatory sites: Na+ and the inhibitory peptide. Removal of the inhibitory peptide perturbs the Na+ site via the α2 helix highlighting the critical role of the α2 helix in regulating ENaC function.

scholarly journals Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT

Science ◽  
2021 ◽  
Vol 372 (6547) ◽  
pp. 1215-1219
Author(s):  
Yiyang Jiang ◽  
Thomas L. Benz ◽  
Stephen B. Long
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Active Site ◽  
Endoplasmic Reticulum Membrane ◽  
Physiochemical Properties ◽  
Amino Terminal ◽  
Cryo Electron Microscopy ◽  
Precursor Proteins ◽  
Hedgehog Proteins ◽  
Hedgehog Acyltransferase

Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo–electron microscopy structures of human HHAT in complex with its palmitoyl–coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.

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