scholarly journals Structural insights into stressosome assembly

IUCrJ ◽  
2019 ◽  
Vol 6 (5) ◽  
pp. 938-947 ◽  
Author(s):  
Eunju Kwon ◽  
Deepak Pathak ◽  
Han-ul Kim ◽  
Pawan Dahal ◽  
Sung Chul Ha ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
Environmental Stress ◽  
Assembly Model ◽  
Bacterial Viability ◽  
Binding Interface ◽  
Cryo Electron Microscopy ◽  
Stress Signals ◽  
Structural Insights

The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA–RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA–RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Å resolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

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

Structure ◽  
2016 ◽  
Vol 24 (4) ◽  
pp. 528-536 ◽  
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

scholarly journals Cryo-EM structures of ASC and NLRC4 CARD filaments reveal a unified mechanism of nucleation and activation of caspase-1

2018 ◽  
Vol 115 (43) ◽  
pp. 10845-10852 ◽  
Author(s):  
Yang Li ◽  
Tian-Min Fu ◽  
Alvin Lu ◽  
Kristen Witt ◽  
Jianbin Ruan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Adaptor Proteins ◽  
Supramolecular Complexes ◽  
Structure Modeling ◽  
Nucleotide Binding Domain ◽  
Caspase 1 ◽  
Cryo Electron Microscopy ◽  
Stress Signals ◽  
Asymmetric Interactions

Canonical inflammasomes are cytosolic supramolecular complexes that activate caspase-1 upon sensing extrinsic microbial invasions and intrinsic sterile stress signals. During inflammasome assembly, adaptor proteins ASC and NLRC4 recruit caspase-1 through homotypic caspase recruitment domain (CARD) interactions, leading to caspase-1 dimerization and activation. Activated caspase-1 processes proinflammatory cytokines and Gasdermin D to induce cytokine maturation and pyroptotic cell death. Here, we present cryo-electron microscopy (cryo-EM) structures of NLRC4 CARD and ASC CARD filaments mediated by conserved three types of asymmetric interactions (types I, II, and III). We find that the CARDs of these two adaptor proteins share a similar assembly pattern, which matches that of the caspase-1 CARD filament whose structure we defined previously. These data indicate a unified mechanism for downstream caspase-1 recruitment through CARD–CARD interactions by both adaptors. Using structure modeling, we further show that full-length NLRC4 assembles via two separate symmetries at its CARD and its nucleotide-binding domain (NBD), respectively.

scholarly journals Cryo-EM structure of the bacterial Ton motor subcomplex ExbB–ExbD provides information on structure and stoichiometry

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Herve Celia ◽  
Istvan Botos ◽  
Xiaodan Ni ◽  
Tara Fox ◽  
Natalia De Val ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
High Resolution ◽  
Outer Membrane ◽  
Motor Function ◽  
Structural Information ◽  
Molecular Motor ◽  
Single Copy ◽  
Proton Motive Force ◽  
Cryo Electron Microscopy

Abstract The TonB–ExbB–ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB–ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

scholarly journals Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy

2019 ◽  
Vol 205 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Hiroki Yamaguchi ◽  
Akiko Kamegawa ◽  
Kunio Nakata ◽  
Tatsuki Kashiwagi ◽  
Toshimi Mizukoshi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Structure ◽  
Leucine Dehydrogenase ◽  
Cryo Electron Microscopy ◽  
Structural Insights

scholarly journals Structural Insights into the BRAF Monomer-to-dimer Transition Mediated by RAS Binding

2021 ◽  
Author(s):  
Juliana Andrea Martinez Fiesco ◽  
David E Durrant ◽  
Deborah K Morrison ◽  
Ping Zhang
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Dynamic Process ◽  
Mammalian Cells ◽  
Dual Function ◽  
Unresolved Issue ◽  
Raf Kinase ◽  
Cryo Electron Microscopy ◽  
Terminal Site ◽  
Structural Insights

An unresolved issue in RAF kinase signaling is how binding of autoinhibited RAF monomers to activated RAS initiates the conformational changes required to form active RAF dimers. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. The RAS binding domain (RBD) of BRAF is resolved in the autoinhibited structures, and we find that neither MEK nor ATP binding is required to stabilize the autoinhibited complexes. Notably, the RBD was found to interact extensively with the 14-3-3 protomer bound to the BRAF C-terminal site. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the 14-3-3 interface have a dual function, first stabilizing RBD orientation in the autoinhibited state and then contributing to full RAS contact.

scholarly journals The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Rebecca L Newcomer ◽  
Jason R Schrad ◽  
Eddie B Gilcrease ◽  
Sherwood R Casjens ◽  
Michael Feig ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Covalent Binding ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Coat Proteins ◽  
Binding Interface ◽  
Cryo Electron Microscopy ◽  
Ob Fold ◽  
Icosahedral Capsid

The major coat proteins of dsDNA tailed phages (order Caudovirales) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary ‘decoration’ (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. The NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.

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.

scholarly journals Structural Insights into TOR Signaling

Genes ◽  
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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