Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

The N-end rule pathway was one of the first ubiquitin (Ub)-dependent degradation pathways to be identified. Ubr1, a single-chain E3 ligase, targets proteins bearing a destabilizing residue at the N-terminus (N-degron) for rapid K48-linked ubiquitination and proteasome-dependent degradation. How Ubr1 catalyses the initiation of ubiquitination on the substrate and elongation of the Ub chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here, we report the cryo-electron microscopy structures of two complexes representing the initiation and elongation intermediates of Ubr1 captured using chemical approaches. In these two structures, Ubr1 adopts different conformations to facilitate the transfer of Ub from Ubc2 to either an N-degron peptide or a monoubiquitinated degron. These structures not only reveal the architecture of the Ubr1 complex but also provide mechanistic insights into the initiation and elongation steps of ubiquitination catalyzed by Ubr1.

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Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus

Journal of Virology ◽

10.1128/jvi.01112-16 ◽

2016 ◽

Vol 90 (21) ◽

pp. 9733-9742 ◽

Cited By ~ 17

Author(s):

Lindsey J. Organtini ◽

Hyunwook Lee ◽

Sho Iketani ◽

Kai Huang ◽

Robert E. Ashley ◽

...

Keyword(s):

Electron Microscopy ◽

Receptor Binding ◽

Conformational Changes ◽

De Novo ◽

Mutational Analysis ◽

Antibody Fragment ◽

Canine Parvovirus ◽

Single Chain ◽

Cryo Electron Microscopy ◽

Resolution Structure

ABSTRACT Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. IMPORTANCE Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time.

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New Structural Insights into the Genome and Minor Capsid Proteins of BK Polyomavirus using Cryo-Electron Microscopy

Structure ◽

10.1016/j.str.2016.02.008 ◽

2016 ◽

Vol 24 (4) ◽

pp. 528-536 ◽

Cited By ~ 32

Author(s):

Daniel L. Hurdiss ◽

Ethan L. Morgan ◽

Rebecca F. Thompson ◽

Emma L. Prescott ◽

Margarita M. Panou ◽

...

Keyword(s):

Electron Microscopy ◽

Capsid Proteins ◽

Cryo Electron Microscopy ◽

Bk Polyomavirus ◽

Structural Insights

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A Human Ubiquitin Conjugating Enzyme (E2)-HECT E3 Ligase Structure-function Screen

Molecular & Cellular Proteomics ◽

10.1074/mcp.o111.013706 ◽

2012 ◽

Vol 11 (8) ◽

pp. 329-341 ◽

Cited By ~ 69

Author(s):

Yi Sheng ◽

Jenny H. Hong ◽

Ryan Doherty ◽

Tharan Srikumar ◽

Jonathan Shloush ◽

...

Keyword(s):

Structure Function ◽

E3 Ligase ◽

Human Ubiquitin ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzyme

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Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a

Journal of Cell Science ◽

10.1242/jcs.258540 ◽

2021 ◽

Author(s):

Yuqing Hou ◽

Lei Zhao ◽

Tomohiro Kubo ◽

Xi Cheng ◽

Nathan McNeill ◽

...

Keyword(s):

Electron Microscopy ◽

Mass Spectrometry ◽

Chlamydomonas Reinhardtii ◽

Model Organism ◽

Super Resolution ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Cryo Electron Microscopy ◽

N Terminus ◽

Central Apparatus

Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a “9+2” axoneme composed of 9 outer doublet microtubules plus 2 central microtubules; the central microtubules together with their projections is termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and ∼36 more are predicted to be CA proteins. Among the candidate CA proteins is the highly conserved FAP70, which also has been reported to be associated with the doublet microtubules. Here we determined by super-resolution structured illumination microscopy that FAP70 is located exclusively in the CA, and show by cryo-electron microscopy that its N-terminus is located at the base of the CA's C2a projection. We also found that fap70-1 mutant axonemes lack most of the C2a projection. Mass spectrometry revealed that fap70-1 axonemes lack not only FAP70 but two other conserved candidate CA proteins, FAP65 and FAP147. Finally, FAP65 and FAP147 co-immunoprecipitated with HA-tagged FAP70. Taken together, these data identify FAP70, FAP65, and FAP147 as the first defining components of the C2a projection.

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The Ubiquitin-conjugating Enzyme (E2) Ube2w Ubiquitinates the N Terminus of Substrates

Journal of Biological Chemistry ◽

10.1074/jbc.c113.477596 ◽

2013 ◽

Vol 288 (26) ◽

pp. 18784-18788 ◽

Cited By ~ 59

Author(s):

Kenneth Matthew Scaglione ◽

Venkatesha Basrur ◽

Naila S. Ashraf ◽

John R. Konen ◽

Kojo S. J. Elenitoba-Johnson ◽

...

Keyword(s):

N Terminus ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzyme

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Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714020343 ◽

2014 ◽

Vol 70 (11) ◽

pp. 2897-2912 ◽

Cited By ~ 19

Author(s):

Ľubomír Borko ◽

Vladena Bauerová-Hlinková ◽

Eva Hostinová ◽

Juraj Gašperík ◽

Konrad Beck ◽

...

Keyword(s):

Amino Acids ◽

Binding Site ◽

Calcium Release ◽

Homology Model ◽

Terminal Region ◽

Phosphoprotein Phosphatase ◽

Cryo Electron Microscopy ◽

Central Helix ◽

N Terminus ◽

Structural Insights

Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals Cαatom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

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Molecular Tectonic Model of Virus Structural Transitions: the Putative Cell Entry States of Poliovirus

Journal of Virology ◽

10.1128/jvi.74.3.1342-1354.2000 ◽

2000 ◽

Vol 74 (3) ◽

pp. 1342-1354 ◽

Cited By ~ 183

Author(s):

David M. Belnap ◽

David J. Filman ◽

Benes L. Trus ◽

Naiqian Cheng ◽

Frank P. Booy ◽

...

Keyword(s):

Electron Microscopy ◽

Infected Cell ◽

Conformational Changes ◽

Viral Protein ◽

Cell Entry ◽

Tectonic Plates ◽

Tectonic Model ◽

Beta Barrel ◽

Cryo Electron Microscopy ◽

N Terminus

ABSTRACT Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ∼22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ∼4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

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Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy

eLife ◽

10.7554/elife.48907 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 43

Author(s):

Ricardo Guerrero-Ferreira ◽

Nicholas MI Taylor ◽

Ana-Andreea Arteni ◽

Pratibha Kumari ◽

Daniel Mona ◽

...

Keyword(s):

Electron Microscopy ◽

Amino Acids ◽

Alpha Synuclein ◽

Fibril Formation ◽

Salt Bridges ◽

Atomic Structures ◽

Cryo Electron Microscopy ◽

N Terminus ◽

Hydrophobic Cleft ◽

Fibril Diameter

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson’s disease (PD). Previously, we reported the structure of alpha-synuclein fibrils (residues 1–121), composed of two protofibrils that are connected via a densely-packed interface formed by residues 50–57 (Guerrero-Ferreira, eLife 218;7:e36402). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.

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Two new polymorphic structures of alpha-synuclein solved by cryo-electron microscopy

10.1101/654582 ◽

2019 ◽

Cited By ~ 4

Author(s):

Ricardo Guerrero-Ferreira ◽

Nicholas M.I. Taylor ◽

Ana-Andrea Arteni ◽

Pratibha Kumari ◽

Daniel Mona ◽

...

Keyword(s):

Electron Microscopy ◽

Amino Acids ◽

Alpha Synuclein ◽

Fibril Formation ◽

Salt Bridges ◽

Atomic Structures ◽

Cryo Electron Microscopy ◽

N Terminus ◽

Hydrophobic Cleft ◽

Fibril Diameter

AbstractIntracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson’s disease (PD). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.Impact statementTwo new polymorphic structures of recombinant human alpha-synuclein fibrils show striking differences to previous structures, while familial PD mutation sites remain crucial for protofilament interaction and fibril stability.

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