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.

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 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 Structural basis for the docking of mTORC1 on the lysosomal surface

Science ◽  
2019 ◽  
Vol 366 (6464) ◽  
pp. 468-475 ◽  
Author(s):  
Kacper B. Rogala ◽  
Xin Gu ◽  
Jibril F. Kedir ◽  
Monther Abu-Remaileh ◽  
Laura F. Bianchi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Growth Factors ◽  
Protein Kinase ◽  
Nucleotide Binding ◽  
Target Of Rapamycin ◽  
Structural Basis ◽  
Mechanistic Target Of Rapamycin ◽  
Rag Gtpases ◽  
Cryo Electron Microscopy ◽  
Guanosine Triphosphatase

The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)–Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP–RagC/D•GDP) permits mTORC1 association. We used cryo–electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor “claw” threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome.

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 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 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 Fission yeast TOR complex 1 phosphorylates Psk1 through evolutionarily conserved interaction mediated by the TOS motif

2021 ◽  
Author(s):  
Yuichi Morozumi ◽  
Ai Hishinuma ◽  
Suguru Furusawa ◽  
Fajar Sofyantoro ◽  
Hisashi Tatebe ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fission Yeast ◽  
Substrate Recognition ◽  
Evolutionary Conservation ◽  
Cellular Growth ◽  
Environmental Cues ◽  
Tor Signaling ◽  
S6 Kinase ◽  
Regulatory Subunits ◽  
Evolutionarily Conserved

TOR complex 1 (TORC1) is a multi-subunit protein kinase complex that controls cellular growth in response to environmental cues. The regulatory subunits of mammalian TORC1 (mTORC1) include RAPTOR, which recruits mTORC1 substrates, such as S6K1 and 4EBP1, by interacting with their TOR signaling (TOS) motif. Despite the evolutionary conservation of TORC1, no TOS motif has been described in lower eukaryotes. Here, we show that the fission yeast S6 kinase Psk1 contains a TOS motif that interacts with Mip1, a RAPTOR ortholog. The TOS motif in Psk1 resembles those in mammals, including the conserved Phe and Asp residues essential for the Mip1 interaction and TORC1-dependent phosphorylation of Psk1. The binding of the TOS motif to Mip1 is dependent on Mip1 Tyr-533, whose equivalent in RAPTOR is known to interact with the TOS motif in their co-crystals. Furthermore, we utilized the mip1-Y533A mutation to screen the known TORC1 substrates in fission yeast and successfully identified Atg13 as a novel TOS motif-containing substrate. These results strongly suggest that the TOS motif represents an evolutionarily conserved mechanism of the substrate recognition by TORC1.

scholarly journals Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

2021 ◽  
Author(s):  
Man Pan ◽  
Qingyun Zheng ◽  
Tian Wang ◽  
Lujun Liang ◽  
Junxiong Mao ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
E3 Ligase ◽  
Degradation Pathways ◽  
Single Chain ◽  
Cryo Electron Microscopy ◽  
N Terminus ◽  
Specific Manner ◽  
Ubiquitin Conjugating Enzyme ◽  
Structural Insights ◽  
Conjugating Enzyme

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.

scholarly journals Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake

2021 ◽  
Vol 7 (29) ◽  
pp. eabg3188
Author(s):  
Miaoqing Hu ◽  
Fan Yang ◽  
Yawen Huang ◽  
Xin You ◽  
Desheng Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cholesterol Level ◽  
Cholesterol Absorption ◽  
Bound State ◽  
Intestinal Cholesterol Absorption ◽  
Cholesterol Uptake ◽  
Cryo Electron Microscopy ◽  
Niemann Pick ◽  
Structural Insights ◽  
Apo State

Niemann-Pick C1-like 1 (NPC1L1) protein plays a central role in the intestinal cholesterol absorption and is the target of a drug, ezetimibe, which inhibits NPC1L1 to reduce cholesterol absorption. Here, we present cryo–electron microscopy structures of human NPC1L1 in apo state, cholesterol-enriched state, and ezetimibe-bound state to reveal molecular details of NPC1L1-mediated cholesterol uptake and ezetimibe inhibition. Comparison of these structures reveals that the sterol-sensing domain (SSD) could respond to the cholesterol level alteration by binding different number of cholesterol molecules. Upon increasing cholesterol level, SSD binds more cholesterol molecules, which, in turn, triggers the formation of a stable structural cluster in SSD, while binding of ezetimibe causes the deformation of the SSD and destroys the structural cluster, leading to the inhibition of NPC1L1 function. These results provide insights into mechanisms of NPC1L1 function and ezetimibe action and are of great significance for the development of new cholesterol absorption inhibitors.

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