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 Lipid-dependent Akt-ivity: where, when, and how

2019 ◽  
Vol 47 (3) ◽  
pp. 897-908 ◽  
Author(s):  
Katharina M. Siess ◽  
Thomas A. Leonard
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Target Of Rapamycin ◽  
Endomembrane System ◽  
Essential Protein ◽  
Mechanistic Target Of Rapamycin ◽  
Subcellular Compartments ◽  
Membrane Bound ◽  
Phosphoinositide 3 Kinase

Abstract Akt is an essential protein kinase activated downstream of phosphoinositide 3-kinase and frequently hyperactivated in cancer. Canonically, Akt is activated by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2, which phosphorylate it on two regulatory residues in its kinase domain upon targeting of Akt to the plasma membrane by PI(3,4,5)P3. Recent evidence, however, has shown that, in addition to phosphorylation, Akt activity is allosterically coupled to the engagement of PI(3,4,5)P3 or PI(3,4)P2 in cellular membranes. Furthermore, the active membrane-bound conformation of Akt is protected from dephosphorylation, and Akt inactivation by phosphatases is rate-limited by its dissociation. Thus, Akt activity is restricted to membranes containing either PI(3,4,5)P3 or PI(3,4)P2. While PI(3,4,5)P3 has long been associated with signaling at the plasma membrane, PI(3,4)P2 is gaining increasing traction as a signaling lipid and has been implicated in controlling Akt activity throughout the endomembrane system. This has clear implications for the phosphorylation of both freely diffusible substrates and those localized to discrete subcellular compartments.

scholarly journals Cryo-EM structure and polymorphism of Aβ amyloid fibrils purified from Alzheimer’s brain tissue

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Marius Kollmer ◽  
William Close ◽  
Leonie Funk ◽  
Jay Rasmussen ◽  
Aref Bsoul ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Brain Tissue ◽  
Amyloid Fibrils ◽  
Structural Basis ◽  
Amyloid Angiopathy ◽  
Cryo Electron Microscopy ◽  
Aβ Amyloid ◽  
Cerebral Amyloid ◽  
Aβ Fibrils

Abstract The formation of Aβ amyloid fibrils is a neuropathological hallmark of Alzheimer’s disease and cerebral amyloid angiopathy. However, the structure of Aβ amyloid fibrils from brain tissue is poorly understood. Here we report the purification of Aβ amyloid fibrils from meningeal Alzheimer’s brain tissue and their structural analysis with cryo-electron microscopy. We show that these fibrils are polymorphic but consist of similarly structured protofilaments. Brain derived Aβ amyloid fibrils are right-hand twisted and their peptide fold differs sharply from previously analyzed Aβ fibrils that were formed in vitro. These data underscore the importance to use patient-derived amyloid fibrils when investigating the structural basis of the disease.

scholarly journals Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association

2016 ◽  
Vol 2 (3) ◽  
pp. e1501502 ◽  
Author(s):  
Thiemo Sprink ◽  
David J. F. Ramrath ◽  
Hiroshi Yamamoto ◽  
Kaori Yamamoto ◽  
Justus Loerke ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Protein Biosynthesis ◽  
Initiation Factor ◽  
Structural Basis ◽  
Initiation Complex ◽  
Cryo Electron Microscopy ◽  
Initiation Factor 2 ◽  
High Resolution Structure ◽  
Guanosine Triphosphatase ◽  
Resolution Structure

Throughout the four phases of protein biosynthesis—initiation, elongation, termination, and recycling—the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAiMet in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation.

How Good Can Single-Particle Cryo-EM Become? What Remains Before It Approaches Its Physical Limits?

2019 ◽  
Vol 48 (1) ◽  
pp. 45-61 ◽  
Author(s):  
Robert M. Glaeser
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Single Particle ◽  
Energy Use ◽  
Optimal Choice ◽  
Structural Basis ◽  
Detective Quantum Efficiency ◽  
Quantum Metrology ◽  
Cryo Electron Microscopy ◽  
Induced Motion

Impressive though the achievements of single-particle cryo–electron microscopy are today, a substantial gap still remains between what is currently accomplished and what is theoretically possible. As is reviewed here, twofold or more improvements are possible as regards ( a) the detective quantum efficiency of cameras at high resolution, ( b) converting phase modulations to intensity modulations in the image, and ( c) recovering the full amount of high-resolution signal in the presence of beam-induced motion of the specimen. In addition, potential for improvement is reviewed for other topics such as optimal choice of electron energy, use of aberration correctors, and quantum metrology. With the help of such improvements, it does not seem to be too much to imagine that determining the structural basis for every aspect of catalytic control, signaling, and regulation, in any type of cell of interest, could easily be accelerated fivefold or more.

scholarly journals Structural basis of actin filament capping at the barbed-end: a cryo-electron microscopy study

2006 ◽  
Vol 25 (23) ◽  
pp. 5626-5633 ◽  
Author(s):  
Akihiro Narita ◽  
Shuichi Takeda ◽  
Atsuko Yamashita ◽  
Yuichiro Maéda
Keyword(s):  
Electron Microscopy ◽  
Actin Filament ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Structural Basis ◽  
Cryo Electron Microscopy

scholarly journals Elucidation of AMPA receptor–stargazin complexes by cryo–electron microscopy

Science ◽  
2016 ◽  
Vol 353 (6294) ◽  
pp. 83-86 ◽  
Author(s):  
Edward C. Twomey ◽  
Maria V. Yelshanskaya ◽  
Robert A. Grassucci ◽  
Joachim Frank ◽  
Alexander I. Sobolevsky
Keyword(s):  
Electron Microscopy ◽  
Cognitive Processes ◽  
Electrostatic Interactions ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Activated State ◽  
Ampar Trafficking ◽  
Excitatory Neurotransmission ◽  
Binding Interfaces ◽  
The Brain

AMPA-subtype ionotropic glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and contribute to high cognitive processes such as learning and memory. In the brain, AMPAR trafficking, gating, and pharmacology is tightly controlled by transmembrane AMPAR regulatory proteins (TARPs). Here, we used cryo–electron microscopy to elucidate the structural basis of AMPAR regulation by one of these auxiliary proteins, TARP γ2, or stargazin (STZ). Our structures illuminate the variable interaction stoichiometry of the AMPAR-TARP complex, with one or two TARP molecules binding one tetrameric AMPAR. Analysis of the AMPAR-STZ binding interfaces suggests that electrostatic interactions between the extracellular domains of AMPAR and STZ play an important role in modulating AMPAR function through contact surfaces that are conserved across AMPARs and TARPs. We propose a model explaining how TARPs stabilize the activated state of AMPARs and how the interactions between AMPARs and their auxiliary proteins control fast excitatory synaptic transmission.

scholarly journals Cryo-EM Reveals the Structural Basis of Microtubule Depolymerization by Kinesin-13s

2017 ◽  
Author(s):  
Matthieu P. M. H. Benoit ◽  
Ana B. Asenjo ◽  
Hernando Sosa
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Distinct Group ◽  
Atomic Resolution ◽  
Structural Basis ◽  
Structural Elements ◽  
Microtubule Depolymerization ◽  
Cryo Electron Microscopy ◽  
Kinesin Superfamily ◽  
Motile Activity

SummaryKinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promotes microtubule depolymerization and lacks motile activity. The molecular mechanism by which the kinesins depolymerize microtubules and are adapted to perform a seemingly very different activity from other kinesins is still unclear. To address this issue we obtained near atomic resolution cryo-electron microscopy (cryo-EM) structures of Drosophila melanogaster kinesin-13 KLP10A constructs bound to curved or straight tubulin in different nucleotide states. The structures show how nucleotide induced conformational changes near the catalytic site are coupled with kinesin-13-specific structural elements to induce tubulin curvature leading to microtubule depolymerization. The data highlight a modular structure that allows similar kinesin core motor-domains to be used for different functions, such as motility or microtubule depolymerization.

scholarly journals On the Structure and Mechanism of Two-Pore Channels

2017 ◽  
Author(s):  
Alexander F. Kintzer ◽  
Robert M. Stroud
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein Kinase ◽  
Channel Structure ◽  
Target Of Rapamycin ◽  
Nutrient Concentrations ◽  
Pore Channel ◽  
Voltage Gated ◽  
Mechanistic Target Of Rapamycin ◽  
Terminal Domain ◽  
Pore Domain

AbstractIn eukaryotes, two-pore channels (TPC1-3) comprise a family of ion channels that regulate the conductance of Na+ and Ca2+ ions across cellular membranes. TPC1-3 form endolysosomal channels, but TPC3 can also function in the plasma membrane. TPC1/3 are voltage-gated channels, but TPC2 opens in response to binding endolysosome-specific lipid phosphatidylinositol-3,5-diphosphate (PI(3,5)P2). Filoviruses, such as Ebola, exploit TPC-mediated ion release as a means of escape from the endolysosome during infection. Antagonists that block TPC1/2 channel conductance abrogate filoviral infections. TPC1/2 form complexes with the mechanistic target of rapamycin complex 1 (mTORC1) at the endolysosomal surface that couple cellular metabolic state and cytosolic nutrient concentrations to the control of membrane potential and pH. We determined the X-ray structure of TPC1 from Arabidopsis thaliana (AtTPC1) to 2.87Å resolution–one of the two first reports of a TPC channel structure. Here we summarize these findings and the implications that the structure may have for understanding endolysosomal control mechanisms and their role in human health.AbbreviationsmTORC1Mechanistic target of rapamycin complex 1TPCTwo-pore channelPI(3,5)P2Phosphatidylinositol-3,5-diphosphateAtTPC1Arabidopsis thaliana TPC1NED19Trans-Ned-19VSDVoltage-sensing domainP1Pore domain in S5-S6P2Pore domain in S11-S12CavVoltage-gated calcium channelNavVoltage-gated sodium channelKvVoltage-gated potassium channelNTDN-terminal domainCTDC-terminal domainEFEF-hand domainNAADPNicotinic acid adenine dinucleotide phosphatePI(4,5)P2Phosphatidylinositol-4,5-diphosphateDHPDihydropyridinePAAPhenylalkylamineBTZBenzothiazepineCaa2+Activating Ca2+-ionCai2+Inhibitory Ca2+-ionfou2Fatty acid oxygenation up-regulated 2SLC38a9Sodium-coupled neutral amino acid transporter 9NPC1Niemann-Pick C1PKAProtein kinase APKCProtein kinase CPKGProtein kinase GH+ATPase - Proton Pump32P– Phosphorus-32

scholarly journals Molecular basis for the ATPase-powered substrate translocation by the Lon AAA+ protease

2020 ◽  
Author(s):  
Kaiming Zhang ◽  
Shanshan Li ◽  
Kan-Yen Hsieh ◽  
Shih-Chieh Su ◽  
Grigore D. Pintilie ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Molecular Basis ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Adenosine Triphosphatases ◽  
Translocation Mechanism ◽  
Atp Binding And Hydrolysis ◽  
Proteolytic Chamber ◽  
Specific Nucleotide

AbstractThe Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of the substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we have determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) at 3.6 Å resolution in a substrate-engaged state. Substrate interactions are mediated by the dual pore-loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states; a closed AAA+ ring is nevertheless maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. The structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.

scholarly journals The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Joseph Atherton ◽  
I-Mei Yu ◽  
Alexander Cook ◽  
Joseph M Muretta ◽  
Agnel Joseph ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Motor Function ◽  
Nucleotide Binding ◽  
Motor Domain ◽  
Kinesin Motor ◽  
Binding Motifs ◽  
Cryo Electron Microscopy ◽  
Specific Sequences

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.

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