RESUMO
The putative Plasmodium translocon of exported proteins (PTEX) is essential for transport of malarial effector proteins across a parasite-encasing vacuolar membrane into host erythrocytes, but the mechanism of this process remains unknown. Here we show that PTEX is a bona fide translocon by determining structures of the PTEX core complex at near-atomic resolution using cryo-electron microscopy. We isolated the endogenous PTEX core complex containing EXP2, PTEX150 and HSP101 from Plasmodium falciparum in the 'engaged' and 'resetting' states of endogenous cargo translocation using epitope tags inserted using the CRISPR-Cas9 system. In the structures, EXP2 and PTEX150 interdigitate to form a static, funnel-shaped pseudo-seven-fold-symmetric protein-conducting channel spanning the vacuolar membrane. The spiral-shaped AAA+ HSP101 hexamer is tethered above this funnel, and undergoes pronounced compaction that allows three of six tyrosine-bearing pore loops lining the HSP101 channel to dissociate from the cargo, resetting the translocon for the next threading cycle. Our work reveals the mechanism of P. falciparum effector export, and will inform structure-based design of drugs targeting this unique translocon.
Assuntos
Microscopia Crioeletrônica , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/ultraestrutura , Animais , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Modelos Biológicos , Modelos Moleculares , Terapia de Alvo Molecular/tendências , Movimento , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Vacúolos/metabolismoRESUMO
Biological membranes define the boundaries of cells and compartmentalize the chemical and physical processes required for life. Many biological processes are carried out by proteins embedded in or associated with such membranes. Determination of membrane protein (MP) structures at atomic or near-atomic resolution plays a vital role in elucidating their structural and functional impact in biology. This endeavor has determined 1198 unique MP structures as of early 2021. The value of these structures is expanded greatly by deposition of their three-dimensional (3D) coordinates into the Protein Data Bank (PDB) after the first atomic MP structure was elucidated in 1985. Since then, free access to MP structures facilitates broader and deeper understanding of MPs, which provides crucial new insights into their biological functions. Here we highlight the structural and functional biology of representative MPs and landmarks in the evolution of new technologies, with insights into key developments influenced by the PDB in magnifying their impact.
Assuntos
Bases de Dados de Proteínas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Bases de Dados de Proteínas/história , História do Século XX , História do Século XXI , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue ("fast gate") known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-ß-synthase (CBS) domains and the intracellular vestibule ("slow gating"). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1-related diseases.
Assuntos
Canais de Cloreto/ultraestrutura , Sequência de Aminoácidos , Membrana Celular/metabolismo , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Microscopia Crioeletrônica/métodos , Humanos , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Modelos MolecularesRESUMO
Electrospray ionization mass spectrometry (ESI-MS) is a ubiquitously used analytical method applied across multiple departments in biopharma, ranging from early research discovery to process development. Accurate, efficient, and consistent protein MS spectral deconvolution across multiple instrument and detector platforms (time-of-flight, Orbitrap, Fourier-transform ion cyclotron resonance) is essential. When proteins are ionized during the ESI process, a distribution of consecutive multiply charged ions are observed on the m/z scale, either positive [M + nH]n+ or negative [M - nH]n- depending on the ionization polarity. The manual calculation of the neutral molecular weight (MW) of single proteins measured by ESI-MS is simple; however, algorithmic deconvolution is required for more complex protein mixtures to derive accurate MWs. Multiple deconvolution algorithms have evolved over the past two decades, all of which have their advantages and disadvantages, in terms of speed, user-input parameters (or ideally lack thereof), and whether they perform optimally on proteins analyzed under denatured or native-MS and solution conditions. Herein, we describe the utility of a parsimonious deconvolution algorithm (explaining the observed spectra with a minimum number of masses) to process a wide range of highly diverse biopharma relevant and research grade proteins and complexes (PEG-GCSF; an IgG1k; IgG1- and IgG2-biotin covalent conjugates; the membrane protein complex AqpZ; a highly polydisperse empty MSP1D1 nanodisc and the tetradecameric chaperone protein complex GroEL) analyzed under native-MS, denaturing LC-MS, and positive and negative modes of ionization, using multiple instruments and therefore multiple data formats. The implementation of a comb filter and peak sharpening option is also demonstrated to be highly effective for deconvolution of highly polydisperse and enhanced separation of a low level lysine glycation post-translational modification (+162.1 Da), partially processed heavy chain lysine residues (+128.1 Da), and loss of N-acetylglucosamine (GlcNAc; -203.1 Da).
Assuntos
Algoritmos , Biofarmácia/métodos , Peso Molecular , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Anticorpos Monoclonais/química , Glicosilação , Imunoconjugados/química , Lisina/química , Proteínas de Membrana/químicaRESUMO
Therapeutic target characterization involves many components, including accurate molecular weight (MW) determination. Knowledge of the accurate MW allows one to detect the presence of post-translational modifications, proteolytic cleavages, and importantly, if the correct construct has been generated and purified. Denaturing liquid chromatography-mass spectrometry (LC-MS) can be an attractive method for obtaining this information. However, membrane protein LC-MS methodology has remained relatively under-explored and under-incorporated in comparison to methods for soluble proteins. Here, systematic investigation of multiple gradients and column chemistries has led to the development of a 5 min denaturing LC-MS method for acquiring membrane protein accurate MW measurements. Conditions were interrogated with membrane proteins, such as GPCRs and ion channels, as well as bispecific antibody constructs of variable sizes with the aim to provide the community with rapid LC-MS methods necessary to obtain chromatographic and accurate MW measurements in a medium- to high-throughput manner. The 5 min method detailed has successfully produced MW measurements for hydrophobic proteins with a wide MW range (17.5 to 105.3 kDa) and provided evidence that some constructs indeed contain unexpected modifications or sequence clipping. This rapid LC-MS method is also capable of baseline separating formylated and nonformylated aquaporinZ membrane protein.
Assuntos
Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Peso MolecularRESUMO
Membrane contact sites (MCS) between organelles are proposed as nexuses for the exchange of lipids, small molecules, and other signals crucial to cellular function and homeostasis. Various protein complexes, such as the endoplasmic reticulum-mitochondrial encounter structure (ERMES), function as dynamic molecular tethers between organelles. Here, we report the reconstitution and characterization of subcomplexes formed by the cytoplasm-exposed synaptotagmin-like mitochondrial lipid-binding protein (SMP) domains present in three of the five ERMES subunits--the soluble protein Mdm12, the endoplasmic reticulum (ER)-resident membrane protein Mmm1, and the mitochondrial membrane protein Mdm34. SMP domains are conserved lipid-binding domains found exclusively in proteins at MCS. We show that the SMP domains of Mdm12 and Mmm1 associate into a tight heterotetramer with equimolecular stoichiometry. Our 17-Å-resolution EM structure of the complex reveals an elongated crescent-shaped particle in which two Mdm12 subunits occupy symmetric but distal positions at the opposite ends of a central ER-anchored Mmm1 homodimer. Rigid body fitting of homology models of these SMP domains in the density maps reveals a distinctive extended tubular structure likely traversed by a hydrophobic tunnel. Furthermore, these two SMP domains bind phospholipids and display a strong preference for phosphatidylcholines, a class of phospholipids whose exchange between the ER and mitochondria is essential. Last, we show that the three SMP-containing ERMES subunits form a ternary complex in which Mdm12 bridges Mmm1 to Mdm34. Our findings highlight roles for SMP domains in ERMES assembly and phospholipid binding and suggest a structure-based mechanism for the facilitated transport of phospholipids between organelles.
Assuntos
Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Sinaptotagminas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Dimerização , Proteínas de Membrana/química , Membranas Mitocondriais/metabolismo , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Membrane contact sites between organelles serve as molecular hubs for the exchange of metabolites and signals. In yeast, the Endoplasmic Reticulum - Mitochondrion Encounter Structure (ERMES) tethers these two organelles likely to facilitate the non-vesicular exchange of essential phospholipids. Present in Fungi and Amoebas but not in Metazoans, ERMES is composed of five distinct subunits; among those, Mdm12, Mmm1 and Mdm34 each contain an SMP domain functioning as a lipid transfer module. We previously showed that the SMP domains of Mdm12 and Mmm1 form a hetero-tetramer. Here we describe our strategy to diversify the number of Mdm12/Mmm1 complexes suited for structural studies. We use sequence analysis of orthologues combined to protein engineering of disordered regions to guide the design of protein constructs and expand the repertoire of Mdm12/Mmm1 complexes more likely to crystallize. Using this combinatorial approach we report crystals of Mdm12/Mmm1 ERMES complexes currently diffracting to 4.5 Å resolution and a new structure of Mdm12 solved at 4.1 Å resolution. Our structure reveals a monomeric form of Mdm12 with a conformationally dynamic N-terminal ß-strand; it differs from a previously reported homodimeric structure where the N-terminal ß strands where swapped to promote dimerization. Based on our electron microscopy data, we propose a refined pseudo-atomic model of the Mdm12/Mmm1 complex that agrees with our crystallographic and small-angle X-ray scattering (SAXS) solution data.
Assuntos
Dictyostelium/química , Retículo Endoplasmático/química , Proteínas Fúngicas/química , Mitocôndrias/química , Proteínas de Protozoários/química , Saccharomyces/química , Modelos Moleculares , Conformação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Survival of the malaria parasite Plasmodium falciparum when it infects red blood cells depends upon its ability to export hundreds of its proteins beyond an encasing vacuole. Protein export is mediated by a parasite-derived protein complex, the Plasmodium translocon of exported proteins (PTEX), and requires unfolding of the different cargos prior to their translocation across the vacuolar membrane. Unfolding is performed by the AAA+protein unfoldase HSP101/ClpB2 and the thioredoxin-2 enzyme (TRX2). Protein trafficking is dramatically impaired in parasites with defective HSP101 or lacking TRX2. These two PTEX subunits drive export and are targets for the design of a novel class of antimalarials: protein export inhibitors. To rationalize inhibitor design, we solved the crystal structure of Pfal-TRX2 at 2.2-Å resolution. Within the asymmetric unit, the three different copies of this protein disulfide reductase sample its two redox catalytic states. Size exclusion chromatography and small-angle X-ray scattering (SAXS) analyses demonstrate that Pfal-TRX2 is monomeric in solution. A non-conserved N-terminal extension precedes the canonical thioredoxin-fold; although it is not observed in our structure, our solution analysis suggests it is flexible in contrast to Plasmodium thioredoxin-1. This represents a first step towards the reconstitution of the entire PTEX for mechanistic and structural studies.
Assuntos
Plasmodium falciparum/química , Proteínas de Protozoários/química , Tiorredoxinas/química , Sequência de Aminoácidos , Animais , Antimaláricos/química , Cromatografia em Gel , Cristalografia por Raios X , Escherichia coli/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Homologia de Sequência de AminoácidosRESUMO
Aberrant folding of proteins in the endoplasmic reticulum activates the bifunctional transmembrane kinase/endoribonuclease Ire1. Ire1 excises an intron from HAC1 messenger RNA in yeasts and Xbp1 messenger RNA in metozoans encoding homologous transcription factors. This non-conventional mRNA splicing event initiates the unfolded protein response, a transcriptional program that relieves the endoplasmic reticulum stress. Here we show that oligomerization is central to Ire1 function and is an intrinsic attribute of its cytosolic domains. We obtained the 3.2-A crystal structure of the oligomer of the Ire1 cytosolic domains in complex with a kinase inhibitor that acts as a potent activator of the Ire1 RNase. The structure reveals a rod-shaped assembly that has no known precedence among kinases. This assembly positions the kinase domain for trans-autophosphorylation, orders the RNase domain, and creates an interaction surface for binding of the mRNA substrate. Activation of Ire1 through oligomerization expands the mechanistic repertoire of kinase-based signalling receptors.
Assuntos
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , Cristalografia por Raios X , Citosol/metabolismo , Ativação Enzimática/efeitos dos fármacos , Íntrons/genética , Glicoproteínas de Membrana/antagonistas & inibidores , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Desnaturação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Ribonucleases/química , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The structure of the protein-translocating channel SecYEß from Pyrococcus furiosus at 3.1-Å resolution suggests a mechanism for chaperoning transmembrane regions of a protein substrate during its lateral delivery into the lipid bilayer. Cytoplasmic segments of SecY orient the C-terminal α-helical region of another molecule, suggesting a general binding mode and a promiscuous guiding surface capable of accommodating diverse nascent chains at the exit of the ribosomal tunnel. To accommodate this putative nascent chain mimic, the cytoplasmic vestibule widens, and a lateral exit portal is opened throughout its entire length for partition of transmembrane helical segments to the lipid bilayer. In this primed channel, the central plug still occludes the pore while the lateral gate is opened, enabling topological arbitration during early protein insertion. In vivo, a 15 amino acid truncation of the cytoplasmic C-terminal helix of SecY fails to rescue a secY-deficient strain, supporting the essential role of this helix as suggested from the structure.
Assuntos
Proteínas de Bactérias/química , Membrana Celular/química , Conformação Proteica , Transporte Proteico/fisiologia , Pyrococcus furiosus/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pyrococcus furiosus/metabolismoRESUMO
BACKGROUND: The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. RESULTS: This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. CONCLUSIONS: Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.
Assuntos
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Clivagem do RNA/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/metabolismo , Glicoproteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , Clivagem do RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
BACKGROUND: Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity. RESULTS: Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal ß-phosphate group of a potent cofactor ADP by sulfur results in ADPßS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn2+ and Cd2+, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors. CONCLUSIONS: Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on protein-protein interactions involving Ire1's kinase domain suggests that protein kinases and pseudokinases encoded in metazoan genomes may use ATP pocket-binding ligands similarly to exert signaling roles other than phosphoryl transfer.
Assuntos
Endorribonucleases/química , Endorribonucleases/metabolismo , Ribonucleases/metabolismo , Cristalografia por Raios X , Endorribonucleases/isolamento & purificação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Ribonucleases/isolamento & purificaçãoRESUMO
Reversed-phase liquid chromatographic mass spectrometry (rpLC-MS) is a universal, platformed, and essential analytical technique within pharmaceutical and biopharmaceutical research. Typical rpLC method gradient times can range from 5 to 20 min. As monoclonal antibody (mAb) therapies continue to evolve and bispecific antibodies (BsAbs) become more established, research stage engineering panels will clearly evolve in size. Therefore, high-throughput (HT) MS and automated deconvolution methods are key for success. Additionally, newer therapeutics such as bispecific T-cell engagers and nucleic acid-based modalities will also require MS characterization. Herein, we present a modality and target agnostic HT solid-phase extraction (SPE) MS method that affords the analysis of a 96-well plate in 41.4 min, compared to the traditional rpLC-MS method that would typically take 14.4 h. The described method can accurately determine the molecular weights for monodispersed and highly polydispersed biotherapeutic species and membrane proteins; determine levels of glycosylation, glycation, and formylation; detect levels of chain mispairing; and determine accurate drug-to-antibody ratio values.
Assuntos
Cromatografia de Fase Reversa , Extração em Fase Sólida , Espectrometria de Massas/métodos , Cromatografia de Fase Reversa/métodos , Peso Molecular , Anticorpos Monoclonais/químicaRESUMO
Eukaryotic cells are characterized by their exquisite compartmentalization resulting from a cornucopia of membrane-bound organelles. Each of these compartments hosts a flurry of biochemical reactions and supports biological functions such as genome storage, membrane protein and lipid biosynthesis/degradation and ATP synthesis, all essential to cellular life. Acting as hubs for the transfer of matter and signals between organelles and throughout the cell, membrane contacts sites (MCSs), sites of close apposition between membranes from different organelles, are essential to cellular homeostasis. One of the now well-acknowledged function of MCSs involves the non-vesicular trafficking of lipids; its characterization answered one long-standing question of eukaryotic cell biology revealing how some organelles receive and distribute their membrane lipids in absence of vesicular trafficking. The endoplasmic reticulum (ER) in synergy with the mitochondria, stands as the nexus for the biosynthesis and distribution of phospholipids (PLs) throughout the cell by contacting nearly all other organelle types. MCSs create and maintain lipid fluxes and gradients essential to the functional asymmetry and polarity of biological membranes throughout the cell. Membrane apposition is mediated by proteinaceous tethers some of which function as lipid transfer proteins (LTPs). We summarize here the current state of mechanistic knowledge of some of the major classes of LTPs and tethers based on the available atomic to near-atomic resolution structures of several "model" MCSs from yeast but also in Metazoans; we describe different models of lipid transfer at MCSs and analyze the determinants of their specificity and directionality. Each of these systems illustrate fundamental principles and mechanisms for the non-vesicular exchange of lipids between eukaryotic membrane-bound organelles essential to a wide range of cellular processes such as at PL biosynthesis and distribution, lipid storage, autophagy and organelle biogenesis.
RESUMO
Signal sequences target proteins for secretion from cells or for integration into cell membranes. As nascent proteins emerge from the ribosome, signal sequences are recognized by the signal recognition particle (SRP), which subsequently associates with its receptor (SR). In this complex, the SRP and SR stimulate each other's GTPase activity, and GTP hydrolysis ensures unidirectional targeting of cargo through a translocation pore in the membrane. To define the mechanism of reciprocal activation, we determined the 1.9 A structure of the complex formed between these two GTPases. The two partners form a quasi-two-fold symmetrical heterodimer. Biochemical analysis supports the importance of the extensive interaction surface. Complex formation aligns the two GTP molecules in a symmetrical, composite active site, and the 3'OH groups are essential for association, reciprocal activation and catalysis. This unique circle of twinned interactions is severed twice on hydrolysis, leading to complex dissociation after cargo delivery.
Assuntos
Proteínas de Bactérias/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Peptídeos/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Dimerização , Ativação Enzimática , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Sinais Direcionadores de Proteínas/fisiologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Ribossomos/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , ThermusRESUMO
Apicomplexans form a large phylum of parasitic protozoa, including the genera Plasmodium, Toxoplasma, and Cryptosporidium, the causative agents of malaria, toxoplasmosis, and cryptosporidiosis, respectively. They cause diseases not only in humans but also in animals, with dramatic consequences in agriculture. Most apicomplexans are vacuole-dwelling and obligate intracellular parasites; as they invade the host cell, they become encased in a parasitophorous vacuole (PV) derived from the host cellular membrane. This creates a parasite-host interface that acts as a protective barrier but also constitutes an obstacle through which the pathogen must import nutrients, eliminate wastes, and eventually break free upon egress. Completion of the parasitic life cycle requires intense remodeling of the infected host cell. Host cell subversion is mediated by a subset of essential effector parasitic proteins and virulence factors actively trafficked across the PV membrane. In the malaria parasite Plasmodium, a unique and highly specialized ATP-driven vacuolar secretion system, the Plasmodium translocon of exported proteins (PTEX), transports effector proteins across the vacuolar membrane. Its core is composed of the three essential proteins EXP2, PTEX150, and HSP101, and is supplemented by the two auxiliary proteins TRX2 and PTEX88. Many but not all secreted malarial effector proteins contain a vacuolar trafficking signal or Plasmodium export element (PEXEL) that requires processing by an endoplasmic reticulum protease, plasmepsin V, for proper export. Because vacuolar parasitic protein export is essential to parasite survival and virulence, this pathway is a promising target for the development of novel antimalarial therapeutics. This review summarizes the current state of structural and mechanistic knowledge on the Plasmodium parasitic vacuolar secretion and effector trafficking pathway, describing its most salient features and discussing the existing differences and commonalities with the vacuolar effector translocation MYR machinery recently described in Toxoplasma and other apicomplexans of significance to medical and veterinary sciences.
RESUMO
Membrane contact sites between the endoplasmic reticulum (ER) and mitochondria function as a central hub for the exchange of phospholipids and calcium. The yeast Endoplasmic Reticulum-Mitochondrion Encounter Structure (ERMES) complex is composed of five subunits that tether the ER and mitochondria. Three ERMES subunits (i.e., Mdm12, Mmm1, and Mdm34) contain the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. The SMP domain belongs to the tubular lipid-binding protein (TULIP) superfamily, which consists of ubiquitous lipid scavenging and transfer proteins. Herein, we describe the methods for expression and purification of recombinant Mdm12, a bona fide SMP-containing protein, together with the subsequent identification of its bound phospholipids by high-performance thin-layer chromatography (HPTLC) and the characterization of its lipid exchange and transfer functions using lipid displacement and liposome flotation in vitro assays with liposomes as model biological membranes. These methods can be applied to the study and characterization of novel lipid-binding and lipid-transfer proteins.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Mitocondriais/metabolismo , Domínios e Motivos de Interação entre Proteínas , Bactérias/genética , Transporte Biológico , Proteínas de Transporte/isolamento & purificação , Cromatografia Líquida , Cromatografia em Camada Fina , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Lipossomos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/isolamento & purificação , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes , Relação Estrutura-Atividade , Leveduras/genéticaRESUMO
In all three kingdoms of life, co-translational targeting of secretory and membrane proteins to the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum is mediated by a ribonucleoprotein complex, the signal recognition particle (SRP), and its membrane-associated receptor (SR). SRP binds to signal sequences of nascent proteins as they emerge from the exit tunnel of the ribosome. The resulting targeting complex, composed of the SRP and the ribosome-nascent chain complex (RNC), then docks with the SR in a GTP-dependent manner. Passing through a complex series of conformational states, SRP and SR deliver the RNC to the translocon, which in turn mediates protein translocation across or integration into the membrane. The core structural and mechanistic principles of SRP-dependent protein targeting are universally conserved. Recent structural investigations combining X-ray crystallography and cryo-electron microscopy have provided new insights into three essentials steps of the SRP-dependent protein targeting cycle: the assembly and interaction of the SRP ribonucleoprotein core, the GTP-dependent SRP-SR association, and the interaction between SRP and the ribosome.
Assuntos
Membrana Celular/química , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/química , Receptores de Peptídeos/química , Partícula de Reconhecimento de Sinal/química , Animais , Proteínas Arqueais/química , Proteínas de Bactérias/química , Sítios de Ligação , Evolução Molecular , Humanos , Ligação Proteica , Conformação Proteica , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
Membrane protein characterization is consistently hampered by challenges with expression, purification, and solubilization. Among several biophysical techniques employed for their characterization, native-mass spectrometry (MS) has emerged as a powerful tool for the analysis of membrane proteins and complexes. Here, two MS platforms, the FT-ICR and Q-ToF, have been explored to analyze the homotetrameric water channel protein, AquaporinZ (AqpZ), under non-denaturing conditions. This 97 kDa membrane protein complex can be readily liberated from the octylglucoside (OG) detergent micelle under a range of instrument conditions on both MS platforms. Increasing the applied collision energy of the FT-ICR collision cell yielded varying degrees of tetramer (97 kDa) liberation from the OG micelles, as well as dissociation into the trimeric (72 kDa) and monomeric (24 kDa) substituents. Tandem-MS on the Q-ToF yielded higher intensity tetramer signal and, depending on the m/z region selected, the observed monomer signal varied in intensity. Precursor ion selection of an m/z range above the expected protein signal distribution, followed by mild collisional activation, is able to efficiently liberate AqpZ with a high S/N ratio. The tetrameric charge state distribution obtained on both instruments demonstrated superpositioning of multiple proteoforms due to varying degrees of N-terminal formylation. Graphical Abstract á .
Assuntos
Espectrometria de Massas/métodos , Proteínas de Membrana/química , Complexos Multiproteicos/química , Aquaporinas/química , Cromatografia Líquida/métodos , Ciclotrons , Detergentes/química , Desenho de Equipamento , Proteínas de Escherichia coli/química , Espectrometria de Massas/instrumentação , Micelas , Desnaturação Proteica , Multimerização Proteica , Espectrometria de Massas em TandemRESUMO
Aquaporins are a family of water and small molecule channels found in organisms ranging from bacteria to animals. One of these channels, the E. coli protein aquaporin Z (AqpZ), has been shown to selectively conduct only water at high rates. We have expressed, purified, crystallized, and solved the X-ray structure of AqpZ. The 2.5 A resolution structure of AqpZ suggests aquaporin selectivity results both from a steric mechanism due to pore size and from specific amino acid substitutions that regulate the preference for a hydrophobic or hydrophilic substrate. This structure provides direct evidence on the molecular mechanisms of specificity between water and glycerol in this family of channels from a single species. It is to our knowledge the first atomic resolution structure of a recombinant aquaporin and so provides a platform for combined genetic, mutational, functional, and structural determinations of the mechanisms of aquaporins and, more generally, the assembly of multimeric membrane proteins.