RESUMEN
The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.
Asunto(s)
Factor de Transcripción AP-1/ultraestructura , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/ultraestructura , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 1 de Ribosilacion-ADP/ultraestructura , Proteínas Adaptadoras del Transporte Vesicular , Antígeno 2 del Estroma de la Médula Ósea/metabolismo , Antígeno 2 del Estroma de la Médula Ósea/ultraestructura , Clatrina , Aparato de Golgi , Células HEK293 , VIH-1 , Humanos , Transporte de Proteínas/fisiología , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
The molecular basis for the severity and rapid spread of the COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04-Å resolution by X-ray crystallography. The structure reveals a â¼60-residue core similar to SARS-CoV-2 ORF7a, with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate noncovalent interface is formed by another SARS-CoV-2-specific sequence, 73YIDI76 Together, the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.
Asunto(s)
Estructura Molecular , SARS-CoV-2/química , Proteínas Virales/química , Evolución Molecular , Evasión InmuneRESUMEN
The accessory protein Nef of human immunodeficiency virus (HIV) is a primary determinant of viral pathogenesis. Nef is abundantly expressed during infection and reroutes a variety of cell surface proteins to disrupt host immunity and promote the viral replication cycle. Nef counteracts host defenses by sequestering and/or degrading its targets via the endocytic and secretory pathways. Nef does this by physically engaging a number of host trafficking proteins. Substantial progress has been achieved in identifying the targets of Nef, and a structural and mechanistic understanding of Nef's ability to command the protein trafficking machinery has recently started to coalesce. Comparative analysis of HIV and simian immunodeficiency virus (SIV) Nef proteins in the context of recent structural advances sheds further light on both viral evolution and the mechanisms whereby trafficking is hijacked. This review describes how advances in cell and structural biology are uncovering in growing detail how Nef subverts the host immune system, facilitates virus release, and enhances viral infectivity.
Asunto(s)
Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Transporte de Proteínas/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Vesículas Cubiertas por Clatrina , Infecciones por VIH/virología , VIH-1/inmunología , Interacciones Huésped-Patógeno , Humanos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación Proteica , Virus de la Inmunodeficiencia de los Simios/inmunología , Proteínas Reguladoras y Accesorias Virales/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A "capture-and-collapse" model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis.
Asunto(s)
Aminoácidos/química , Antígenos Bacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Portadoras/química , Fibrinógeno/química , Streptococcus pyogenes/química , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fibrinógeno/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.
Asunto(s)
Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/metabolismo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/enzimología , Animales , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Membrana Celular/microbiología , Biología Computacional , Exotoxinas/metabolismo , Femenino , Prueba de Complementación Genética , Histidina Quinasa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Datos de Secuencia Molecular , Neutrófilos/microbiología , Mutación Puntual , Polisacárido Liasas/metabolismo , Polisacáridos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , VirulenciaRESUMEN
The assembly of the autophagy initiation machinery nucleates autophagosome biogenesis, including in the PINK1- and Parkin-dependent mitophagy pathway implicated in Parkinson's disease. The structural interaction between the sole transmembrane autophagy protein, autophagy-related protein 9A (ATG9A), and components of the Unc-51-like autophagy activating kinase (ULK1) complex is one of the major missing links needed to complete a structural map of autophagy initiation. We determined the 2.4-Å x-ray crystallographic structure of the ternary structure of ATG9A carboxyl-terminal tail bound to the ATG13:ATG101 Hop1/Rev7/Mad2 (HORMA) dimer, which is part of the ULK1 complex. We term the interacting portion of the extreme carboxyl-terminal part of the ATG9A tail the "HORMA dimer-interacting region" (HDIR). This structure shows that the HDIR binds to the HORMA domain of ATG101 by ß sheet complementation such that the ATG9A tail resides in a deep cleft at the ATG13:ATG101 interface. Disruption of this complex in cells impairs damage-induced PINK1/Parkin mitophagy mediated by the cargo receptor NDP52.
Asunto(s)
Proteínas de la Membrana , Mitofagia , Proteínas Relacionadas con la Autofagia/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas Quinasas/genéticaRESUMEN
The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.
Asunto(s)
Biliverdina , Colorantes , Proteínas Luminiscentes/genética , Ingeniería , Proteína Fluorescente RojaRESUMEN
Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other.
Asunto(s)
Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Autofagosomas/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/química , Proteínas Relacionadas con la Autofagia/genética , Cristalografía , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Proteínas de Unión a Fosfato/química , Proteínas de Unión a Fosfato/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Mutación Puntual , Conformación Proteica en Hélice alfa , Transporte de Proteínas , Transducción de Señal , Relación Estructura-ActividadRESUMEN
The molecular basis for the severity and rapid spread of the COVID-19 disease caused by SARS-CoV-2 is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04 Å resolution by x-ray crystallography. The structure reveals a ~60 residue core similar to SARS-CoV ORF7a with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate non-covalent interface is formed by another SARS-CoV-2-specific sequence, 73 YIDI 76 . Together the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.
RESUMEN
Tetherin is a host defense factor that physically prevents virion release from the plasma membrane. The Nef accessory protein of simian immunodeficiency virus (SIV) engages the clathrin adaptor AP-2 to downregulate tetherin via its DIWK motif. As human tetherin lacks DIWK, antagonism of tetherin by Nef is a barrier to simian-human transmission of non-human primate lentiviruses. To determine the molecular basis for tetherin counteraction, we reconstituted the AP-2 complex with a simian tetherin and SIV Nef and determined its structure by cryoelectron microscopy (cryo-EM). Nef refolds the first α-helix of the ß2 subunit of AP-2 to a ß hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of most other Nef substrates, including MHC class I, CD3, and CD4 but overlaps with the site for the restriction factor SERINC5. This structure explains the dependence of SIVs on tetherin DIWK and consequent barrier to human transmission.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Antígeno 2 del Estroma de la Médula Ósea/química , Antígeno 2 del Estroma de la Médula Ósea/farmacología , Infecciones por Lentivirus/prevención & control , Infecciones por Lentivirus/transmisión , Zoonosis/virología , Complejo 2 de Proteína Adaptadora/química , Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades beta de Complejo de Proteína Adaptadora/química , Animales , Sitios de Unión , Complejo CD3/metabolismo , Antígenos CD4/metabolismo , Membrana Celular/efectos de los fármacos , Microscopía por Crioelectrón , Regulación hacia Abajo , Productos del Gen nef/química , Productos del Gen nef/metabolismo , Células HEK293 , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Infecciones por Lentivirus/virología , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Cultivo Primario de Células , Conformación Proteica , Conformación Proteica en Hélice alfa , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Virus de la Inmunodeficiencia de los Simios/metabolismo , Virión/efectos de los fármacosRESUMEN
This corrects the article DOI: 10.1038/nmicrobiol.2016.155.
RESUMEN
No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently â¼90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant 'reading head' in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M-C4BP interaction, and also inform a path towards vaccine design.