RESUMEN
The Wnt signaling pathway controls numerous cell fates in animal development and is also a major cancer pathway. Dishevelled (Dvl) transduces the Wnt signal by interacting with the cytoplasmic Axin complex. Dvl and Axin each contain a DIX domain whose molecular properties and structure are unknown. Here, we demonstrate that the DIX domain of Dvl2 mediates dynamic polymerization, which is essential for the signaling activity of Dvl2. The purified domain polymerizes gradually, reversibly and in a concentration dependent manner, ultimately forming fibrils. The Axin DIX domain has a novel structural fold largely composed of beta-strands that engage in head-to-tail self-interaction to form filaments in the crystal. The DIX domain thus seems to mediate the formation of a dynamic interaction platform with a high local concentration of binding sites for transient Wnt signaling partners; this represents a previously uncharacterized mechanistic principle, signaling by reversible polymerization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Modelos Moleculares , Fosfoproteínas/metabolismo , Polímeros/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Proteína Axina , Secuencia de Bases , Cristalización , Proteínas Dishevelled , Humanos , Inmunoprecipitación , Microscopía Electrónica , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , UltracentrifugaciónRESUMEN
Endophilins have been proposed to have an enzymatic activity (a lysophosphatidic acid acyl transferase or LPAAT activity) that can make phosphatidic acid in membranes. This activity is thought to change the bilayer asymmetry in such a way that negative membrane curvature at the neck of a budding vesicle will be stabilized. An LPAAT activity has also been proposed for CtBP/BARS (carboxy-terminal binding protein/brefeldin A-ribosylated substrate), a transcription co-repressor that is implicated in dynamin-independent endocytosis and fission of the Golgi in mitosis. Here we show that the LPAAT activity associated with endophilin is a contaminant of the purification procedure and can be also found associated with the pleckstrin homology domain of dynamin. Likewise, the LPAAT activity associated with CtBP/BARS is also a co-purification artefact. The proposed locus of activity in endophilins includes the BAR domain, which has no catalytic site but instead senses positive membrane curvature. These data will prompt a re-evaluation of the molecular details of membrane budding.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Endocitosis , Aparato de Golgi/metabolismo , Fosfoproteínas/metabolismo , Aciltransferasas/química , Aciltransferasas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Oxidorreductasas de Alcohol , Animales , Artefactos , Bovinos , Membrana Celular/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Membranas Intracelulares/metabolismo , Lisofosfolípidos/metabolismo , Ratones , Mitosis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/genética , Estructura Terciaria de Proteína , Ratas , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
NF-kappaB activation occurs upon degradation of its inhibitor I-kappaB and requires prior phosphorylation of the inhibitor by I-kappaB kinase (IKK). Activity of IKK is governed by its noncatalytic subunit IKKgamma. Signaling defects due to missense mutations in IKKgamma have been correlated to its inability to either become ubiquitylated or bind ubiquitin noncovalently. Because the relative contribution of these events to signaling had remained unknown, we have studied mutations in the coil-zipper (CoZi) domain of IKKgamma that either impair signaling or cause constitutive NF-kappaB activity. Certain signaling-deficient alleles neither bound ubiquitin nor were they ubiquitylated by TRAF6. Introducing an activating mutation into those signaling-impaired alleles restored their ubiquitylation and created mutants constitutively activating NF-kappaB without repairing the ubiquitin-binding defect. Constitutive activity therefore arises downstream of ubiquitin binding but upstream of ubiquitylation. Such constitutive activity reveals a signal-processing function for IKKgamma beyond that of a mere ubiquitin-binding adaptor. We propose that this signal processing may involve homophilic CoZi interactions as suggested by the enhanced affinity of CoZi domains from constitutively active IKKgamma.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/fisiología , Quinasa I-kappa B/química , Quinasa I-kappa B/fisiología , Leucina Zippers/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Alelos , Animales , Línea Celular , Línea Celular Tumoral , Regulación hacia Abajo/genética , Activación Enzimática/genética , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Células Jurkat , Leucina Zippers/genética , Ratones , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Ratas , Transducción de Señal/genética , Transducción de Señal/fisiología , Ubiquitina/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Proteínas Portadoras/aislamiento & purificación , Clatrina/metabolismo , Células Eucariotas/metabolismo , Factor de Transcripción AP-1/metabolismo , Vesículas Transportadoras/metabolismo , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Sitios de Unión/genética , Células COS , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Compartimento Celular/fisiología , Vesículas Cubiertas por Clatrina/genética , Vesículas Cubiertas por Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/ultraestructura , Endosomas/genética , Endosomas/metabolismo , Endosomas/ultraestructura , Células Eucariotas/citología , Datos de Secuencia Molecular , Mutación/fisiología , Unión Proteica/fisiología , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/genética , Vesículas Transportadoras/ultraestructura , Red trans-Golgi/genética , Red trans-Golgi/metabolismo , Red trans-Golgi/ultraestructuraRESUMEN
Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents differ only by the length of the alkyl chain, the contribution of the detergent micelle to the hydrodynamic volume is variable, whereas the contribution of the protein is constant. By using this approach, several parameters of membrane proteins can be estimated by extrapolation, such as the radius at the midpoint of the membrane, the average radius, the Stokes radius, and the excluded volume. The molecular mass of the protein can be determined by two independent measurements that arise from the behaviour of the free detergent micelle and protein-detergent micelle during size exclusion chromatography and the determination of the detergent-protein ratio. Determining the dimensions of protein-detergent micelles may facilitate membrane protein purification and crystallization by defining the accessibility of the protein surface.
Asunto(s)
Cromatografía en Gel/métodos , Proteínas de la Membrana/química , Translocasas Mitocondriales de ADP y ATP/química , Peso Molecular , Animales , Bovinos , Fenómenos Químicos , Detergentes/química , Detergentes/farmacología , Glucósidos/química , Glucósidos/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/aislamiento & purificación , Micelas , Translocasas Mitocondriales de ADP y ATP/aislamiento & purificación , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
A spectrum of membrane curvatures exists within cells, and proteins have evolved different modules to detect, create, and maintain these curvatures. Here we present the crystal structure of one such module found within human FCHo2. This F-BAR (extended FCH) module consists of two F-BAR domains, forming an intrinsically curved all-helical antiparallel dimer with a Kd of 2.5 microM. The module binds liposomes via a concave face, deforming them into tubules with variable diameters of up to 130 nm. Pulse EPR studies showed the membrane-bound dimer is the same as the crystal dimer, although the N-terminal helix changed conformation on membrane binding. Mutation of a phenylalanine on this helix partially attenuated narrow tubule formation, and resulted in a gain of curvature sensitivity. This structure shows a distant relationship to curvature-sensing BAR modules, and suggests how similar coiled-coil architectures in the BAR superfamily have evolved to expand the repertoire of membrane-sculpting possibilities.
Asunto(s)
Membrana Celular/química , Modelos Moleculares , Proteínas/química , Secuencia de Aminoácidos , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Unión a Ácidos Grasos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/química , Proteínas de la Membrana , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaAsunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Membranas Artificiales , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Calorimetría , Proteínas Portadoras/ultraestructura , Dinaminas/química , Células HeLa , Humanos , Hidrólisis , Liposomas/química , Liposomas/metabolismo , Liposomas/ultraestructura , Ratones , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Rayos XRESUMEN
Filamins are essential in cell motility and many developmental processes. They are large actin cross linking proteins that contain actin binding domains in their N termini and a long rod region constructed from 24 tandem Ig domains. Dimerization is crucial for the actin crosslinking function of filamins and requires the most C-terminal Ig domain. We describe here the crystal structure of this 24th Ig domain (Ig24) of human filamin C and show how it mediates dimerization. The dimer interface is novel and quite different to that seen in the Dictyostelium discoideum filamin analog. The sequence signature of the dimerization interface suggests that the C-terminal domains of all vertebrate filamins share the same dimerization mechanism. Furthermore, we show that point mutations in the dimerization interface disrupt the dimer and that the dissociation constant for recombinant Ig24 is in the micromolar range.
Asunto(s)
Proteínas Contráctiles/química , Proteínas Contráctiles/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Vertebrados , Actinas/química , Actinas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Ácido Aspártico/metabolismo , Cromatografía en Gel , Dicroismo Circular , Proteínas Contráctiles/genética , Reactivos de Enlaces Cruzados/química , Cristalografía por Rayos X , Dictyostelium/química , Dimerización , Filaminas , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Microfilamentos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , UltracentrifugaciónRESUMEN
Subunit c of the H(+) transporting ATP synthase is an essential part of its membrane domain that participates in transmembrane proton conduction. The annular architecture of the subunit c from different species has been previously reported. However, little is known about the type of interactions that affect the formation of c-rings in the ATPase complex. Here we report that subunit c over-expressed in Escherichia coli and purified in non-ionic detergent solutions self-assembles into annular structures in the absence of other subunits of the complex. The results suggest that the ability of subunit c to form rings is determined by its primary structure.
Asunto(s)
ATPasas de Translocación de Protón Bacterianas/química , Escherichia coli/enzimología , Subunidades de Proteína , ATPasas de Translocación de Protón Bacterianas/aislamiento & purificación , ATPasas de Translocación de Protón Bacterianas/ultraestructura , Detergentes/química , Dimetilaminas/química , Escherichia coli/química , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica , Peso Molecular , Unión Proteica/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/ultraestructura , Ultracentrifugación/métodosRESUMEN
Hepatitis B virus consists of an icosahedral core containing the double-stranded DNA genome, enveloped by a membrane with embedded surface proteins. The crystal structure of the core protein has been solved but little information about the structure of the surface proteins has so far been available. There are three sizes of surface protein, small (S), medium (M) and large (L), which form disulfide-bonded homo- and heterodimers. The three proteins, expressed from different start sites in the coding sequence, share the common C-terminal S region; the M protein contains an additional preS2 sequence N-terminal to S, and the L protein a further preS1 sequence N-terminal to M. In infected individuals, the surface proteins are produced in huge excess over the amount needed for viral envelopment and are secreted as a heterogeneous mixture of isometric and tubular subviral particles. We have used electron cryomicroscopy to study tubular particles extracted from human serum. Helical Fourier-Bessel analysis was used to calculate a low-resolution map, although it showed that the tubes were quite disordered. From the symmetry derived from this analysis, we used single-particle methods to improve the resolution. We found that the tubes had a diameter of approximately 250 A, with spike-like features projecting from the membrane. In the plane of the membrane the proteins appear to be close packed. We propose a model for the packing arrangement of surface protein dimers in the tubes.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/química , Virus de la Hepatitis B/química , Estructura Cuaternaria de Proteína , Microscopía por Crioelectrón , Dimerización , Virus de la Hepatitis B/aislamiento & purificación , Humanos , Procesamiento de Imagen Asistido por Computador , Modelos Moleculares , Suero/virologíaRESUMEN
The tyrosine kinase Met, the product of the c-met proto-oncogene and the receptor for hepatocyte growth factor/scatter factor (HGF/SF), mediates signals critical for cell survival and migration. The human pathogen Listeria monocytogenes exploits Met signaling for invasion of host cells via its surface protein InlB. We present the crystal structure of the complex between a large fragment of the human Met ectodomain and the Met-binding domain of InlB. The concave face of the InlB leucine-rich repeat region interacts tightly with the first immunoglobulin-like domain of the Met stalk, a domain which does not bind HGF/SF. A second contact between InlB and the Met Sema domain locks the otherwise flexible receptor in a rigid, signaling competent conformation. Full Met activation requires the additional C-terminal domains of InlB which induce heparin-mediated receptor clustering and potent signaling. Thus, although it elicits a similar cellular response, InlB is not a structural mimic of HGF/SF.
Asunto(s)
Proteínas Bacterianas/química , Listeria monocytogenes/química , Proteínas de la Membrana/química , Proteínas Proto-Oncogénicas c-met/química , Sitios de Unión , Heparina/farmacología , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Proteínas Repetidas Ricas en Leucina , Modelos Biológicos , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/metabolismo , Proto-Oncogenes MasRESUMEN
Mitochondrial carriers are believed widely to be homodimers both in the inner membrane of the organelle and in detergents. The dimensions and molecular masses of the detergent and protein-detergent micelles were measured for yeast ADP/ATP carriers in a range of different detergents. The radius of the carrier at the midpoint of the membrane, its average radius, its Stokes' radius, its molecular mass, and its excluded volume were determined. These parameters are consistent with the known structural model of the bovine ADP/ATP carrier and they demonstrate that the yeast mitochondrial ADP/ATP carriers are monomeric in detergents. Therefore, models of substrate transport have to be considered in which the carrier operates as a monomer rather than as a dimer.
Asunto(s)
Detergentes , Translocasas Mitocondriales de ADP y ATP/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efectos de los fármacos , Animales , Bovinos , Cromatografía en Gel , Micelas , Translocasas Mitocondriales de ADP y ATP/aislamiento & purificación , Peso MolecularRESUMEN
Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin-A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N-BAR domains function as low-affinity dimers regulating binding partner recruitment to areas of high membrane curvature.
Asunto(s)
Aciltransferasas/química , Aciltransferasas/metabolismo , Membrana Celular/química , Aciltransferasas/ultraestructura , Secuencia de Aminoácidos , Animales , Dimerización , Humanos , Liposomas/química , Fusión de Membrana , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Electricidad EstáticaRESUMEN
Hepatitis B virus, a widespread and serious human pathogen, replicates by reverse transcription of an RNA intermediate. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. Using electron cryomicroscopy, we compare the structures of the bacterially expressed RNA-containing core particle and the mature DNA-containing core particle extracted from virions. We show that the mature core contains 240 subunits in a T = 4 arrangement similar to that in expressed core (T is the triangulation number and the icosahedral shell contains 60 T subunits). During the infective cycle, the core assembles in an immature state around a complex of viral pregenomic RNA and polymerase. After reverse transcription with concomitant degradation of the RNA, the now mature core buds through a cellular membrane containing the surface proteins to become enveloped. Envelopment must not happen before reverse transcription is completed, so it has been hypothesized that a change in capsid structure may signal maturation. Our results show significant differences in structure between the RNA- and DNA-containing cores. One such difference is in a hydrophobic pocket, formed largely from residues that, on mutation, lead to abnormal secretion. We suggest that the changes we see are related to maturation and control of envelopment, and we propose a mechanism based on DNA synthesis for their triggering.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/química , Virus de la Hepatitis B/química , Modelos Moleculares , Ensamble de Virus , Microscopía por Crioelectrón , ADN Viral/biosíntesis , Virus de la Hepatitis B/fisiología , Humanos , ARN Viral/metabolismo , Transcripción Reversa , Virión/químicaRESUMEN
Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The plasmid partitioning loci encode two proteins, ParA and ParB, and a cis-acting centromere-like site denoted parS. The chromosomally encoded homologues of ParA and ParB, Soj and Spo0J, play an active role in chromosome segregation during bacterial cell division and sporulation. Spo0J is a DNA-binding protein that binds to parS sites in vivo. We have solved the X-ray crystal structure of a C-terminally truncated Spo0J (amino acids 1-222) from Thermus thermophilus to 2.3 A resolution by multiwavelength anomalous dispersion. It is a DNA-binding protein with structural similarity to the helix-turn-helix (HTH) motif of the lambda repressor DNA-binding domain. The crystal structure is an antiparallel dimer with the recognition alpha-helices of the HTH motifs of each monomer separated by a distance of 34 A corresponding to the length of the helical repeat of B-DNA. Sedimentation velocity and equilibrium ultracentrifugation studies show that full-length Spo0J exists in a monomer-dimer equilibrium in solution and that Spo0J1-222 is exclusively monomeric. Sedimentation of the C-terminal domain of Spo0J shows it to be exclusively dimeric, confirming that the C-terminus is the primary dimerization domain. We hypothesize that the C-terminus mediates dimerization of Spo0J, thereby effectively increasing the local concentration of the N-termini, which most probably dimerize, as shown by our structure, upon binding to a cognate parS site.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Thermus thermophilus , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Centrifugación por Gradiente de Densidad , Segregación Cromosómica , Secuencia Conservada , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Dimerización , Secuencias Hélice-Giro-Hélice , Modelos Moleculares , Peso Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Thermus thermophilus/genética , Thermus thermophilus/metabolismoRESUMEN
In Saccharomyces cerevisiae, at least three proteins (IF(1), STF(1), and STF(2)) appear to be involved in the regulation of ATP synthase. Both IF(1) and STF(1) inhibit F(1), whereas the proposed function for STF(2) is to facilitate the binding of IF(1) and STF(1) to F(1). The oligomerization properties of yeast IF(1) and STF(1) have been investigated by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that IF(1) and STF(1) oligomerize in opposite directions in relation to pH, suggesting that both proteins might regulate yeast F(1)F(0)-ATPase under different conditions. Their effects on bovine F-ATPases are also described. Whereas bovine IF(1) inhibits yeast F(1)-ATPase even better than yeast IF(1) or STF(1), the capability of yeast IF(1) to inhibit the bovine enzyme is very low and decreases with time. Such an effect is also observed in the study of the homologous inhibition of yeast F(1)-ATPase. Yeast inhibitors are not as effective as their bovine counterpart, and the complex seems to dissociate gradually.
Asunto(s)
Proteínas Fúngicas/fisiología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Animales , Bovinos , Proteínas Fúngicas/química , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , ATPasas de Translocación de Protón/metabolismoRESUMEN
The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Vesículas Cubiertas/metabolismo , Proteínas del Citoesqueleto , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Liposomas/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Estructura Terciaria de Proteína , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Secuencia de Aminoácidos , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Vesículas Cubiertas/química , Cristalografía por Rayos X , Dimerización , Drosophila/química , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Liposomas/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Little is known about the large ectodomain of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor (HGF/SF). Here, we establish by deletion mutagenesis that the HGF/SF and heparin-binding sites of MET are contained within a large N-terminal domain spanning the alpha-chain (amino acids 25-307) and the first 212 amino acids of the beta-chain (amino acids 308-519). Neither the cystine-rich domain (amino acids 520-561) nor the C-terminal half of MET (amino acids 562-932) bind HGF/SF or heparin directly. The MET ectodomain, which behaves as a rod-shaped monomer with a large Stokes radius in solution, binds HGF/SF in the absence or presence of heparin, and forms a stable HGF/SF-heparin-MET complex with 1:1:1 stoichiometry. We also show that the ligand-binding domain adopts a beta-propeller fold, which is similar to the N-terminal domain of alphaV integrin, and that the C-terminal half contains four Ig domains (amino acids 563-654, 657-738, 742-836, and 839-924) of the unusual structural E set, which could be modeled on bacterial enzymes. Our studies provide 3D models and a functional map of the MET ectodomain. They have broad implications for structure-function of the MET receptor and the related semaphorin and plexin proteins.