Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

AbstractThe lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a–GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

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Membrane characteristics tune activities of endosomal and autophagic human VPS34 complexes

eLife ◽

10.7554/elife.58281 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Yohei Ohashi ◽

Shirley Tremel ◽

Glenn Robert Masson ◽

Lauren McGinney ◽

Jerome Boulanger ◽

...

Keyword(s):

Complex I ◽

Minor Role ◽

Structural Determinants ◽

Lipid Kinase ◽

Complex Ii ◽

Exchange Mass Spectrometry ◽

Specific Complex ◽

Endocytic Sorting ◽

Hydrogen Deuterium ◽

A Minor

The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.

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Structural pathway for allosteric activation of the autophagic PI 3-kinase complex I

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911612116 ◽

2019 ◽

Vol 116 (43) ◽

pp. 21508-21513 ◽

Cited By ~ 7

Author(s):

Lindsey N. Young ◽

Felix Goerdeler ◽

James H. Hurley

Keyword(s):

Protein Kinase ◽

Complex I ◽

Structural Changes ◽

Kinase Domain ◽

Activation Loop ◽

Class Iii ◽

Lipid Kinase ◽

Protein Kinase Domain ◽

Autophagy Induction ◽

Enzymatic Activation

Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.

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Structural Pathway for Allosteric Activation of the Autophagic PI 3-Kinase Complex I

10.1101/758797 ◽

2019 ◽

Author(s):

Lindsey N. Young ◽

Felix Goerdeler ◽

James H. Hurley

Keyword(s):

Protein Kinase ◽

Complex I ◽

Structural Changes ◽

Kinase Domain ◽

Activation Loop ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Kinase ◽

Autophagy Induction ◽

Enzymatic Activation

AbstractAutophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the pro-autophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryo-EM was used to visualize stepwise PI3KC3-C1 activating effects of binding the NRFB2 MIT domains. Binding of a single NRBF2 MIT domain to bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.

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Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

eLife ◽

10.7554/elife.05115 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 79

Author(s):

Sulochanadevi Baskaran ◽

Lars-Anders Carlson ◽

Goran Stjepanovic ◽

Lindsey N Young ◽

Do Jin Kim ◽

...

Keyword(s):

Complex I ◽

Kinase Activity ◽

Kinase Domain ◽

Scaffolding Protein ◽

Hydrogen Deuterium Exchange ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Kinase ◽

Hydrogen Deuterium ◽

Phosphatidylinositol 3

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

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Faculty Opinions recommendation of Functional modules and structural basis of conformational coupling in mitochondrial complex I.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.3838956.4838055 ◽

2010 ◽

Author(s):

Thomas Meier

Keyword(s):

Complex I ◽

Structural Basis ◽

Mitochondrial Complex I ◽

Functional Modules ◽

Mitochondrial Complex ◽

Conformational Coupling

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ZBP1 promotes LPS-induced cell death and IL-1β release via RHIM-mediated interactions with RIPK1

Nature Communications ◽

10.1038/s41467-020-20357-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hayley I. Muendlein ◽

Wilson M. Connolly ◽

Zoie Magri ◽

Irina Smirnova ◽

Vladimir Ilyukha ◽

...

Keyword(s):

Immune Response ◽

Cell Death ◽

Complex Formation ◽

Complex I ◽

Yersinia Pseudotuberculosis ◽

Caspase 8 ◽

Inflammasome Activation ◽

Inflammatory Signaling ◽

Complex Ii ◽

Dependent Model

AbstractInflammation and cell death are closely linked arms of the host immune response to infection, which when carefully balanced ensure host survival. One example of this balance is the tightly regulated transition from TNFR1-associated pro-inflammatory complex I to pro-death complex II. By contrast, here we show that a TRIF-dependent complex containing FADD, RIPK1 and caspase-8 (that we have termed the TRIFosome) mediates cell death in response to Yersinia pseudotuberculosis and LPS. Furthermore, we show that constitutive binding between ZBP1 and RIPK1 is essential for the initiation of TRIFosome interactions, caspase-8-mediated cell death and inflammasome activation, thus positioning ZBP1 as an effector of cell death in the context of bacterial blockade of pro-inflammatory signaling. Additionally, our findings offer an alternative to the TNFR1-dependent model of complex II assembly, by demonstrating pro-death complex formation reliant on TRIF signaling.

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In situ structure and organization of the influenza C virus surface glycoprotein

Nature Communications ◽

10.1038/s41467-021-21818-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Steinar Halldorsson ◽

Kasim Sader ◽

Jack Turner ◽

Lesley J. Calder ◽

Peter B. Rosenthal

Keyword(s):

Membrane Fusion ◽

Hexagonal Lattice ◽

Surface Glycoprotein ◽

Structural Basis ◽

Virus Surface ◽

The Matrix ◽

Influenza C Virus ◽

Subtomogram Averaging ◽

Electron Cryotomography

AbstractThe lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice. The splaying of these domains is also coupled to changes in the structure of the stem region which is involved in membrane fusion, thereby linking HEF’s membrane fusion conformation with its assembly on the virus surface. The glycoprotein lattice can form independent of other virion components but we show a major role for the matrix layer in particle formation.

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Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization

Nature Communications ◽

10.1038/s41467-021-25248-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christopher Agnew ◽

Pelin Ayaz ◽

Risa Kashima ◽

Hanna S. Loving ◽

Prajakta Ghatpande ◽

...

Keyword(s):

Md Simulations ◽

Kinase Domain ◽

Structural Basis ◽

Type I ◽

Type Ii ◽

Exchange Mass Spectrometry ◽

Receptor Complexes ◽

Bmp Receptors ◽

Morphogenetic Protein ◽

Smad Proteins

AbstractUpon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

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Structural basis for a complex I mutation that blocks pathological ROS production

Nature Communications ◽

10.1038/s41467-021-20942-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhan Yin ◽

Nils Burger ◽

Duvaraka Kula-Alwar ◽

Dunja Aksentijević ◽

Hannah R. Bridges ◽

...

Keyword(s):

Point Mutation ◽

Complex I ◽

Structural Changes ◽

Ischemia Reperfusion ◽

Single Point ◽

Structural Basis ◽

Single Point Mutation ◽

Ros Production

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

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