RESUMO
Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Nonconventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here, we present a comparative study of the heptahelical membrane protein bacteriorhodopsin in detergent micelles, amphipols, and nanodiscs. Our results confirm that nonconventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are, in principle, accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguish regions of bacteriorhodopsin that mediate membrane/solvent contacts in the tested environments, whereas the protein's functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies.
Assuntos
Bacteriorodopsinas/química , Escherichia coli/química , Halobacterium salinarum/química , Proteínas de Membrana/química , Membranas Artificiais , Frações Subcelulares/metabolismo , Bacteriorodopsinas/genética , Bacteriorodopsinas/metabolismo , Meios de Cultura , Detergentes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Halobacterium salinarum/genética , Halobacterium salinarum/metabolismo , Bicamadas Lipídicas/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Micelas , Modelos Moleculares , Mimetismo Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfolipídeos/química , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Frações Subcelulares/químicaRESUMO
The shared cytokine receptor gp130 signals as a homodimer or heterodimer through activation of Janus kinases (Jaks) associated with the receptor intracellular domains. Here, we reconstitute, in parts and whole, the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6Rα) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to form a continuous connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three lobed structure comprising FERM-SH2, pseudokinase, and kinase modules possessing extensive intersegmental flexibility that likely facilitates allosteric activation. Single-particle imaging of the gp130/IL-6/IL-6Rα/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. Jak1 association with gp130 is enhanced by the presence of a membrane environment.
Assuntos
Receptor gp130 de Citocina/química , Janus Quinase 1/química , Proteínas Recombinantes de Fusão/química , 1,2-Dipalmitoilfosfatidilcolina/química , Humanos , Microscopia Eletrônica , Modelos Moleculares , Nanoestruturas , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.
Assuntos
Proteína de Domínio de Morte Associada a Fas/química , Mutação , Receptor fas/química , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Síndrome Linfoproliferativa Autoimune/genética , Caspase 8/química , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas/genética , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Receptor fas/genéticaRESUMO
Binding of the 4-1BB ligand (4-1BBL) to its receptor, 4-1BB, provides the T lymphocyte with co-stimulatory signals for survival, proliferation, and differentiation. Importantly, the 4-1BB-4-1BBL pathway is a well known target for anti-cancer immunotherapy. Here we present the 2.3-A crystal structure of the extracellular domain of human 4-1BBL. The ectodomain forms a homotrimer with an extended, three-bladed propeller structure that differs from trimers formed by other members of the tumor necrosis factor (TNF) superfamily. Based on the 4-1BBL structure, we modeled its complex with 4-1BB, which was consistent with images obtained by electron microscopy, and verified the binding site by site-directed mutagenesis. This structural information will facilitate the development of immunotherapeutics targeting 4-1BB.
Assuntos
Ligante 4-1BB/química , Ligante 4-1BB/fisiologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/fisiologia , Ligante 4-1BB/genética , Sítios de Ligação , Proliferação de Células , Clonagem Molecular , Cristalografia por Raios X/métodos , Dimerização , Citometria de Fluxo , Humanos , Microscopia Eletrônica/métodos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/químicaRESUMO
Biophysical studies of membrane proteins are often impeded by the requirement for a membrane mimicking environment. Detergent micelles are the most common choice, but the denaturing properties make them unsatisfactory for studies of many membrane proteins and their interactions. In the present work, we explore phospholipid bilayer nanodiscs as membrane mimics and employ electron microscopy and solution NMR spectroscopy to characterize the structure and function of the human voltage dependent anion channel (VDAC-1) as an example of a polytopic integral membrane protein. Electron microscopy reveals the formation of VDAC-1 multimers, an observation that is consistent with results obtained in native mitochondrial outer membranes. High-resolution NMR spectroscopy demonstrates a well folded VDAC-1 protein and native NADH binding functionality. The observed chemical shift changes upon addition of the native ligand NADH to nanodisc-embedded VDAC-1 resemble those of micelle-embedded VDAC-1, indicating a similar structure and function in the two membrane-mimicking environments. Overall, the ability to study integral membrane proteins at atomic resolution with solution NMR in phospholipid bilayers, rather than in detergent micelles, offers exciting novel possibilities to approach the biophysical properties of membrane proteins under nondenaturing conditions, which makes this technology particular suitable for protein-protein interactions and other functional studies.
Assuntos
Bicamadas Lipídicas/química , Nanoestruturas/química , Canal de Ânion 1 Dependente de Voltagem/química , Dimiristoilfosfatidilcolina/química , Humanos , Tamanho da Partícula , Conformação Proteica , Propriedades de SuperfícieRESUMO
Urea transporters (UTs) facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. Bacteria use urea as a means to survive in acidic environments and/or as a nitrogen source. The UT from Actinobacillus pleuropneumoniae, ApUT, the pathogen that causes porcine pleurisy and pneumonia, was expressed in Escherichia coli and purified. Analysis of the recombinant protein using cross-linking and blue-native gel electrophoresis established that ApUT is a dimer in detergent solution. Purified protein was reconstituted into proteoliposomes and urea efflux was measured by stopped-flow fluorometry to determine the urea transport kinetics of ApUT. The measured urea flux was saturable, could be inhibited by phloretin, and was not affected by pH. Two-dimensional crystals of the biologically active ApUT show that it is also dimeric in a lipid membrane and provide the first structural information on a member of the UT family.
Assuntos
Actinobacillus pleuropneumoniae/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Estrutura Quaternária de Proteína , Actinobacillus pleuropneumoniae/química , Animais , Proteínas de Bactérias/genética , Cristalização , Detergentes/química , Dimerização , Humanos , Proteínas de Membrana Transportadoras/genética , Permeabilidade , Suínos , Ureia/metabolismo , Transportadores de UreiaRESUMO
Human phospholamban (PLN), a 30 kDa homopentamer in the sarcoplasmic reticulum (SR) membrane, controls the magnitude of heart muscle contraction and relaxation by regulating the calcium pumping activity of the SR Ca(2+)-ATPase (SERCA). When PLN is not phosphorylated, it binds and inhibits SERCA. Phosphorylation of PLN at S16 or T17 releases such inhibitory effect. It remains a matter of debate whether phosphorylation perturbs the structure of PLN, which in turn affects its interaction with SERCA. Here we examine by NMR spectroscopy the structure and dynamics of PLN pentamer with a physiologically relevant, phosphorylation-mimicking mutation, S16E. Based on extensive NMR data, including NOEs, dipolar couplings, and solvent exchange of backbone amides, we conclude that the phosphorylation-mimicking mutation does not perturb the pentamer structure. However, (15)N R(1) and R(2) relaxation rates and (15)N((1)H) NOEs suggest subtle differences in the dynamics of the extramembrane portion of the protein.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Substituição de Aminoácidos , Proteínas de Ligação ao Cálcio/genética , Humanos , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Serina/metabolismo , Relação Estrutura-AtividadeRESUMO
We report an application of the principle of multivalency to create new antimicrobial agents using the reactive polymaleic anhydride (PMA) chain to link antimicrobial tetrapeptides to afford multivalent variants containing approximately 40 monomer units. Relative to the free peptides, the product shows a 10-fold improvement in IC(50) without provoking more severe hemolysis of red blood cells. Thus, multivalency or polyvalency may offer a route to enhance the activity of antimicrobial peptides.