Structure of BipA in GTP form bound to the ratcheted ribosome

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.

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

Science Advances ◽

10.1126/sciadv.abb0147 ◽

2020 ◽

Vol 6 (33) ◽

pp. eabb0147 ◽

Cited By ~ 2

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.

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

10.1101/2021.05.18.444632 ◽

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.

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Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

Science ◽

10.1126/science.1259724 ◽

2015 ◽

Vol 347 (6217) ◽

pp. 75-78 ◽

Cited By ~ 154

Author(s):

Peter S. Shen ◽

Joseph Park ◽

Yidan Qin ◽

Xueming Li ◽

Krishna Parsawar ◽

...

Keyword(s):

Electron Microscopy ◽

Transfer Rna ◽

Exit Channel ◽

Ribosomal Subunits ◽

Cryo Electron Microscopy ◽

Nascent Chain ◽

New Gene ◽

Carboxy Terminal ◽

A Site ◽

Control Complex

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo–electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions (“CAT tails”).

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Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State

Journal of Virology ◽

10.1128/jvi.01443-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 10

Author(s):

Mike Strauss ◽

Lise Schotte ◽

Krishanthi S. Karunatilaka ◽

David J. Filman ◽

James M. Hogle

Keyword(s):

Electron Microscopy ◽

Careful Analysis ◽

Productive Infection ◽

Altered State ◽

Virus Structure ◽

Cryo Electron Microscopy ◽

A Cell ◽

Single Domain Antibodies ◽

A Site ◽

Polypeptide Chains

ABSTRACT By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding permitted a more detailed view of the virus structure than was previously possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered and which become rearranged. The higher resolution of these structures also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, which, in retrospect, were present in lower-resolution structures but not recognized. These structural observations help to explain preexisting mutational data and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion. IMPORTANCE When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection and how these changes in the virus lead to productive-infection events.

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Heterodimers as the Structural Unit of the T=1 Capsid of the Fungal Double-Stranded RNA Rosellinia necatrix Quadrivirus 1

Journal of Virology ◽

10.1128/jvi.01013-16 ◽

2016 ◽

Vol 90 (24) ◽

pp. 11220-11230 ◽

Cited By ~ 14

Author(s):

Daniel Luque ◽

Carlos P. Mata ◽

Fernando González-Camacho ◽

José M. González ◽

Josué Gómez-Blanco ◽

...

Keyword(s):

Electron Microscopy ◽

Molecular Sieves ◽

Three Dimensional ◽

Rosellinia Necatrix ◽

Double Stranded Rna ◽

Content Type ◽

Cryo Electron Microscopy ◽

Wide Range ◽

The Family ◽

Dsrna Viruses

ABSTRACTMost double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the familyQuadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ∼450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization.IMPORTANCEGiven their importance to health, members of the familyReoviridaeare the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungusRosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

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

PLoS ONE ◽

10.1371/journal.pone.0252497 ◽

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.

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Cryo-electron Microscopy Study of the Genome Release of the Dicistrovirus Israeli Acute Bee Paralysis Virus

Journal of Virology ◽

10.1128/jvi.02060-16 ◽

2016 ◽

Vol 91 (4) ◽

Cited By ~ 7

Author(s):

Edukondalu Mullapudi ◽

Tibor Füzik ◽

Antonín Přidal ◽

Pavel Plevka

Keyword(s):

Electron Microscopy ◽

The United States ◽

Capsid Proteins ◽

Content Type ◽

Cryo Electron Microscopy ◽

The Family ◽

Electron Microscopy Analysis ◽

Colony Collapse ◽

Acute Bee Paralysis Virus ◽

Paralysis Virus

ABSTRACT Viruses of the family Dicistroviridae can cause substantial economic damage by infecting agriculturally important insects. Israeli acute bee paralysis virus (IAPV) causes honeybee colony collapse disorder in the United States. High-resolution molecular details of the genome delivery mechanism of dicistroviruses are unknown. Here we present a cryo-electron microscopy analysis of IAPV virions induced to release their genomes in vitro. We determined structures of full IAPV virions primed to release their genomes to a resolution of 3.3 Å and of empty capsids to a resolution of 3.9 Å. We show that IAPV does not form expanded A particles before genome release as in the case of related enteroviruses of the family Picornaviridae. The structural changes observed in the empty IAPV particles include detachment of the VP4 minor capsid proteins from the inner face of the capsid and partial loss of the structure of the N-terminal arms of the VP2 capsid proteins. Unlike the case for many picornaviruses, the empty particles of IAPV are not expanded relative to the native virions and do not contain pores in their capsids that might serve as channels for genome release. Therefore, rearrangement of a unique region of the capsid is probably required for IAPV genome release. IMPORTANCE Honeybee populations in Europe and North America are declining due to pressure from pathogens, including viruses. Israeli acute bee paralysis virus (IAPV), a member of the family Dicistroviridae, causes honeybee colony collapse disorder in the United States. The delivery of virus genomes into host cells is necessary for the initiation of infection. Here we present a structural cryo-electron microscopy analysis of IAPV particles induced to release their genomes. We show that genome release is not preceded by an expansion of IAPV virions as in the case of related picornaviruses that infect vertebrates. Furthermore, minor capsid proteins detach from the capsid upon genome release. The genome leaves behind empty particles that have compact protein shells.

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

10.1101/2021.03.13.435256 ◽

2021 ◽

Cited By ~ 2

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.

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

eLife ◽

10.7554/elife.59038 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

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.

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Structural Insights into TOR Signaling

Genes ◽

10.3390/genes11080885 ◽

2020 ◽

Vol 11 (8) ◽

pp. 885

Author(s):

Lucas Tafur ◽

Jennifer Kefauver ◽

Robbie Loewith

Keyword(s):

Electron Microscopy ◽

Protein Kinase ◽

Molecular Biology ◽

Cellular Responses ◽

Therapeutic Strategies ◽

Cellular Growth ◽

Tor Signaling ◽

Cellular Homeostasis ◽

Cryo Electron Microscopy ◽

Structural Insights

The Target of Rapamycin (TOR) is a highly conserved serine/threonine protein kinase that performs essential roles in the control of cellular growth and metabolism. TOR acts in two distinct multiprotein complexes, TORC1 and TORC2 (mTORC1 and mTORC2 in humans), which maintain different aspects of cellular homeostasis and orchestrate the cellular responses to diverse environmental challenges. Interest in understanding TOR signaling is further motivated by observations that link aberrant TOR signaling to a variety of diseases, ranging from epilepsy to cancer. In the last few years, driven in large part by recent advances in cryo-electron microscopy, there has been an explosion of available structures of (m)TORC1 and its regulators, as well as several (m)TORC2 structures, derived from both yeast and mammals. In this review, we highlight and summarize the main findings from these reports and discuss both the fascinating and unexpected molecular biology revealed and how this knowledge will potentially contribute to new therapeutic strategies to manipulate signaling through these clinically relevant pathways.

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