RESUMEN
Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes. Autophagy-related proteins (ATGs) 2A and 9A have an essential role in autophagy by mediating lipid transfer and re-equilibration between membranes for autophagosome formation. Here we report the cryo-electron microscopy structures of human ATG2A in complex with WD-repeat protein interacting with phosphoinositides 4 (WIPI4) at 3.2 Å and the ATG2A-WIPI4-ATG9A complex at 7 Å global resolution. On the basis of molecular dynamics simulations, we propose a mechanism of lipid extraction from the donor membranes. Our analysis revealed 3:1 stoichiometry of the ATG9A-ATG2A complex, directly aligning the ATG9A lateral pore with ATG2A lipid transfer cavity, and an interaction of the ATG9A trimer with both the N-terminal and the C-terminal tip of rod-shaped ATG2A. Cryo-electron tomography of ATG2A liposome-binding states showed that ATG2A tethers lipid vesicles at different orientations. In summary, this study provides a molecular basis for the growth of the phagophore membrane and lends structural insights into spatially coupled lipid transport and re-equilibration during autophagosome formation.
RESUMEN
The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence with four telomeric repeats (Q4), resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI-binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures under physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is an excellent EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.
Asunto(s)
Berberina , G-Cuádruplex , Berberina/química , Berberina/análogos & derivados , Berberina/farmacología , Humanos , ADN/química , Ácidos Nucleicos de Péptidos/químicaRESUMEN
AMBRA1 is a tumor suppressor protein that functions as a substrate receptor of the ubiquitin conjugation system with roles in autophagy and the cell cycle regulatory network. The intrinsic disorder of AMBRA1 has thus far precluded its structural determination. To solve this problem, we analyzed the dynamics of AMBRA1 using hydrogen deuterium exchange mass spectrometry (HDX-MS). The HDX results indicated that AMBRA1 is a highly flexible protein and can be stabilized upon interaction with DDB1, the adaptor of the Cullin4A/B E3 ligase. Here, we present the cryo-EM structure of AMBRA1 in complex with DDB1 at 3.08 Å resolution. The structure shows that parts of the N- and C-terminal structural regions in AMBRA1 fold together into the highly dynamic WD40 domain and reveals how DDB1 engages with AMBRA1 to create a binding scaffold for substrate recruitment. The N-terminal helix-loop-helix motif and WD40 domain of AMBRA1 associate with the double-propeller fold of DDB1. We also demonstrate that DDB1 binding-defective AMBRA1 mutants prevent ubiquitination of the substrate Cyclin D1 in vitro and increase cell cycle progression. Together, these results provide structural insights into the AMBRA1-ubiquitin ligase complex and suggest a mechanism by which AMBRA1 acts as a hub involved in various physiological processes.
Asunto(s)
Proteínas Portadoras , Proteínas de Unión al ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Portadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Puntos de Control del Ciclo Celular , Ubiquitina/metabolismoRESUMEN
CULLIN-RING ligases constitute the largest group of E3 ubiquitin ligases. While some CULLIN family members recruit adapters before engaging further with different substrate receptors, homo-dimeric BTB-Kelch family proteins combine adapter and substrate receptor into a single polypeptide for the CULLIN3 family. However, the entire structural assembly and molecular details have not been elucidated to date. Here, we present a cryo-EM structure of the CULLIN3RBX1 in complex with Kelch-like protein 22 (KLHL22) and a mitochondrial glutamate dehydrogenase complex I (GDH1) at 3.06 Å resolution. The structure adopts a W-shaped architecture formed by E3 ligase dimers. Three CULLIN3KLHL22-RBX1 dimers were found to be dynamically associated with a single GDH1 hexamer. CULLIN3KLHL22-RBX1 ligase mediated the polyubiquitination of GDH1 in vitro. Together, these results enabled the establishment of a structural model for understanding the complete assembly of BTB-Kelch proteins with CULLIN3 and how together they recognize oligomeric substrates and target them for ubiquitination.
Asunto(s)
Proteínas Cullin , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Cullin/metabolismo , Unión Proteica , Microscopía por Crioelectrón , Estructura Terciaria de Proteína , Proteínas Portadoras/metabolismo , UbiquitinaciónRESUMEN
Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide (NMN) during axonal injury. Its activation mechanism is not fully understood. Here, we generate a nanobody, Nb-C6, that specifically recognizes NMN-activated SARM1. Nb-C6 stains only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN, and partially activates SARM1 in vitro and in cells. Cryo-EM of NMN/SARM1/Nb-C6 complex shows an octameric structure with ARM domains bending significantly inward and swinging out together with TIR domains. Nb-C6 binds to SAM domain of the activated SARM1 and stabilized its ARM domain. Mass spectrometry analyses indicate that the activated SARM1 in solution is highly dynamic and that the neighboring TIRs form transient dimers via the surface close to one BB loop. We show that Nb-C6 is a valuable tool for studies of SARM1 activation.
Asunto(s)
Axones , Mononucleótido de Nicotinamida , Mononucleótido de Nicotinamida/metabolismo , Axones/metabolismo , Dominios Proteicos , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismoRESUMEN
Autophagy is a conserved eukaryotic pathway critical for cellular adaptation to changes in nutrition levels and stress. The class III phosphatidylinositol (PI)3-kinase complexes I and II (PI3KC3-C1 and -C2) are essential for autophagosome initiation and maturation, respectively, from highly curved vesicles. We used a cell-free reaction that reproduces a key autophagy initiation step, LC3 lipidation, as a biochemical readout to probe the role of autophagy-related gene (ATG)14, a PI3KC3-C1-specific subunit implicated in targeting the complex to autophagy initiation sites. We reconstituted LC3 lipidation with recombinant PI3KC3-C1, -C2, or various mutant derivatives added to extracts derived from a CRISPR/Cas9-generated ATG14-knockout cell line. Both complexes C1 and C2 require the C-terminal helix of VPS34 for activity on highly curved membranes. However, only complex C1 supports LC3 lipidation through the curvature-targeting amphipathic lipid packing sensor (ALPS) motif of ATG14. Furthermore, the ALPS motif and VPS34 catalytic activity are required for downstream recruitment of WD-repeat domain phosphoinositide-interacting protein (WIPI)2, a protein that binds phosphatidylinositol 3-phosphate and its product phosphatidylinositol 3, 5-bisphosphate, and a WIPI-binding protein, ATG2A, but do not affect membrane association of ATG3 and ATG16L1, enzymes contributing directly to LC3 lipidation. These data reveal the nuanced role of the ATG14 ALPS in membrane curvature sensing, suggesting that the ALPS has additional roles in supporting LC3 lipidation.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Autofagia/fisiología , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras , Células HEK293 , Humanos , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first ß sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA ß sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 ß sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.
Asunto(s)
Autofagia , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas Relacionadas con la Autofagia , Beclina-1/química , Beclina-1/genética , Sitios de Unión , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/genética , Microscopía por Crioelectrón , Activación Enzimática , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Simulación de Dinámica Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Relación Estructura-Actividad , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The Escherichia coli signal recognition particle (SRP) receptor, FtsY, plays a fundamental role in co-translational targeting of membrane proteins via the SRP pathway. Efficient targeting relies on membrane interaction of FtsY and heterodimerization with the SRP protein Ffh, which is driven by detachment of α helix (αN1) in FtsY. Here we show that apart from the heterodimer, FtsY forms a nucleotide-dependent homodimer on the membrane, and upon αN1 removal also in solution. Homodimerization triggers reciprocal stimulation of GTP hydrolysis and occurs in vivo. Biochemical characterization together with integrative modeling suggests that the homodimer employs the same interface as the heterodimer. Structure determination of FtsY NG+1 with GMPPNP shows that a dimerization-induced conformational switch of the γ-phosphate is conserved in Escherichia coli, filling an important gap in SRP GTPase activation. Our findings add to the current understanding of SRP GTPases and may challenge previous studies that did not consider homodimerization of FtsY.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Sitios de Unión , Membrana Celular/química , Proteínas de Escherichia coli/metabolismo , Guanosina Trifosfato/química , Hidrólisis , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
Transcriptionally repressive histone H3 lysine 27 methylation by Polycomb repressive complex 2 (PRC2) is essential for cellular differentiation and development. Here we report cryo-electron microscopy structures of human PRC2 in a basal state and two distinct active states while in complex with its cofactors JARID2 and AEBP2. Both cofactors mimic the binding of histone H3 tails. JARID2, methylated by PRC2, mimics a methylated H3 tail to stimulate PRC2 activity, whereas AEBP2 interacts with the RBAP48 subunit, mimicking an unmodified H3 tail. SUZ12 interacts with all other subunits within the assembly and thus contributes to the stability of the complex. Our analysis defines the complete architecture of a functionally relevant PRC2 and provides a structural framework to understand its regulation by cofactors, histone tails, and RNA.
Asunto(s)
Complejo Represivo Polycomb 2/química , Proteínas Represoras/química , Microscopía por Crioelectrón , Histonas/química , Humanos , Metilación , Complejo Represivo Polycomb 2/ultraestructura , Unión Proteica , Conformación Proteica , Proteínas Represoras/ultraestructuraRESUMEN
Pex1 and Pex6 form a heterohexameric motor essential for peroxisome biogenesis and function, and mutations in these AAA-ATPases cause most peroxisome-biogenesis disorders in humans. The tail-anchored protein Pex15 recruits Pex1/Pex6 to the peroxisomal membrane, where it performs an unknown function required for matrix-protein import. Here we determine that Pex1/Pex6 from S. cerevisiae is a protein translocase that unfolds Pex15 in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Our structural studies of Pex15 in isolation and in complex with Pex1/Pex6 illustrate that Pex15 binds the N-terminal domains of Pex6, before its C-terminal disordered region engages with the pore loops of the motor, which then processively threads Pex15 through the central pore. Furthermore, Pex15 directly binds the cargo receptor Pex5, linking Pex1/Pex6 to other components of the peroxisomal import machinery. Our results thus support a role of Pex1/Pex6 in mechanical unfolding of peroxins or their extraction from the peroxisomal membrane during matrix-protein import.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de la Membrana/metabolismo , Peroxisomas/enzimología , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Conformación Proteica , Saccharomyces cerevisiaeRESUMEN
The class III PI 3-kinase, VPS34 forms distinct complexes essential for cargo sorting and membrane trafficking in endocytosis as well as for autophagosome nucleation and maturation. We used integrative structural biology approach to provide insights into the conformational dynamics of the complex and mechanisms that regulate VPS34 activity at the membrane.
RESUMEN
The lysosomal membrane is the locus for sensing cellular nutrient levels, which are transduced to mTORC1 via the Rag GTPases and the Ragulator complex. The crystal structure of the five-subunit human Ragulator at 1.4 Å resolution was determined. Lamtor1 wraps around the other four subunits to stabilize the assembly. The Lamtor2:Lamtor3 dimer stacks upon Lamtor4:Lamtor5 to create a platform for Rag binding. Hydrogen-deuterium exchange was used to map the Rag binding site to the outer face of the Lamtor2:Lamtor3 dimer and to the N-terminal intrinsically disordered region of Lamtor1. EM was used to reconstruct the assembly of the full-length RagAGTP:RagCGDP dimer bound to Ragulator at 16 Å resolution, revealing that the G-domains of the Rags project away from the Ragulator core. The combined structural model shows how Ragulator functions as a platform for the presentation of active Rags for mTORC1 recruitment, and might suggest an unconventional mechanism for Rag GEF activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Diana Mecanicista del Complejo 1 de la Rapamicina/química , Proteínas de Unión al GTP Monoméricas/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Microscopía Electrónica , Simulación del Acoplamiento Molecular , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is required for the initiation of essentially all macroautophagic processes. PI3KC3-C1 consists of the lipid kinase catalytic subunit VPS34, the VPS15 scaffold, and the regulatory BECN1 and ATG14 subunits. The VPS34 catalytic domain and BECN1:ATG14 subcomplex do not touch, and it is unclear how allosteric signals are transmitted to VPS34. We used EM and crosslinking mass spectrometry to dissect five conformational substates of the complex, including one in which the VPS34 catalytic domain is dislodged from the complex but remains tethered by an intrinsically disordered linker. A "leashed" construct prevented dislodging without interfering with the other conformations, blocked enzyme activity in vitro, and blocked autophagy induction in yeast cells. This pinpoints the dislodging and tethering of the VPS34 catalytic domain, and its regulation by VPS15, as a master allosteric switch in autophagy induction.
Asunto(s)
Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Regulación Alostérica , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/genética , Células HEK293 , Humanos , Espectrometría de Masas/métodos , Mutación , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Relación Estructura-Actividad , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismoRESUMEN
The intrinsically disordered scaffold proteins AFF1/4 and the transcription elongation factors ELL1/2 are core components of the super elongation complex required for HIV-1 proviral transcription. Here we report the 2.0-Å resolution crystal structure of the human ELL2 C-terminal domain bound to its 50-residue binding site on AFF4, the ELLBow. The ELL2 domain has the same arch-shaped fold as the tight junction protein occludin. The ELLBow consists of an N-terminal helix followed by an extended hairpin that we refer to as the elbow joint, and occupies most of the concave surface of ELL2. This surface is important for the ability of ELL2 to promote HIV-1 Tat-mediated proviral transcription. The AFF4-ELL2 interface is imperfectly packed, leaving a cavity suggestive of a potential binding site for transcription-promoting small molecules.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/genética , VIH-1/fisiología , Provirus/fisiología , Proteínas Represoras/química , Elongación de la Transcripción Genética/fisiología , Factores de Elongación Transcripcional/química , Síndrome de Inmunodeficiencia Adquirida/virología , Sitios de Unión/genética , Sistemas CRISPR-Cas , Cristalografía por Rayos X , Regulación Viral de la Expresión Génica , Técnicas de Inactivación de Genes , VIH-1/patogenicidad , Células HeLa , Humanos , Células Jurkat , Mutagénesis , Unión Proteica/genética , Dominios Proteicos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismo , Activación Viral/genética , Latencia del Virus/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn2+-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop. The structure and functional assays collectively support an integrative structure and a bipartite binding model, wherein the TAR central loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat ARM.
Asunto(s)
Ciclina T/química , Quinasa 9 Dependiente de la Ciclina/química , ADN Viral/química , Duplicado del Terminal Largo de VIH , Proteínas Represoras/química , Factores de Elongación Transcripcional/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , ADN Viral/metabolismo , Medición de Intercambio de Deuterio , VIH-1/genética , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Provirus/genética , Proteínas Represoras/metabolismo , Dispersión del Ángulo Pequeño , Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The ULK1 complex, consisting of the ULK1 protein kinase itself, FIP200, Atg13, and Atg101, controls the initiation of autophagy in animals. We determined the structure of the complex of the human Atg13 HORMA (Hop1, Rev7, Mad2) domain in complex with the full-length HORMA domain-only protein Atg101. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer and the S. pombe Atg13-Atg101 HORMA complex. The WF finger motif that is essential for function in human Atg101 is sequestered in a hydrophobic pocket, suggesting that the exposure of this motif is regulated. Benzamidine molecules from the crystallization solution mark two hydrophobic pockets that are conserved in, and unique to, animals, and are suggestive of sites that could interact with other proteins. These features suggest that the activity of the animal Atg13-Atg101 subcomplex is regulated and that it is an interaction hub for multiple partners.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Autofagia/genética , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Mad2/química , Proteínas Serina-Treonina Quinasas/química , Proteínas de Transporte Vesicular/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Benzamidinas/química , Sitios de Unión , Cristalografía por Rayos X , Expresión Génica , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Alineación de Secuencia , Células Sf9 , Spodoptera , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The AAA+ ATPase Vps4 disassembles ESCRT-III and is essential for HIV-1 budding and other pathways. Vps4 is a paradigmatic member of a class of hexameric AAA+ ATPases that disassemble protein complexes without degradation. To distinguish between local displacement versus global unfolding mechanisms for complex disassembly, we carried out hydrogen/deuterium exchange during Saccharomyces cerevisiae Vps4 disassembly of a chimeric Vps24-2 ESCRT-III filament. EX1 exchange behavior shows that Vps4 completely unfolds ESCRT-III substrates on a time scale consistent with the disassembly reaction. The established unfoldase ClpX showed the same pattern, thus demonstrating a common unfolding mechanism. Vps4 hexamers containing a single cysteine residue in the pore loops were cross-linked to ESCRT-III subunits containing unique cysteines within the folded core domain. These data support a mechanism in which Vps4 disassembles its substrates by completely unfolding them and threading them through the central pore.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Pliegue de Proteína , Transporte de ProteínasRESUMEN
The Atg1 complex, comprising Atg1, Atg13, Atg17, Atg29, and Atg31, is a key initiator of autophagy. The Atg17-Atg31-Atg29 subcomplex is constitutively present at the phagophore assembly site (PAS), while Atg1 and Atg13 join the complex when autophagy is triggered by starvation or other signals. We sought to understand the energetics and dynamics of assembly using isothermal titration calorimetry (ITC), sedimentation velocity analytical ultracentrifugation, and hydrogen-deuterium exchange (HDX). We showed that the membrane and Atg13-binding domain of Atg1, Atg1EAT, is dynamic on its own, but is rigidified in its high-affinity (â¼100 nM) complex with Atg13. Atg1EAT and Atg13 form a 2:2 dimeric assembly and together associate with lower affinity (â¼10 µM) with the 2:2:2 Atg17-Atg31-Atg29 complex. These results lead to an overall model for the assembly pathway of the Atg1 complex. The model highlights the Atg13-Atg17 binding event as the weakest link in the assembly process and thus as a natural regulatory checkpoint.
Asunto(s)
Autofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/química , Modelos Biológicos , Unión ProteicaRESUMEN
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.