RESUMEN
SignalP ( https://services.healthtech.dtu.dk/services/SignalP-6.0/ ) is a very popular prediction method for signal peptides, the intrinsic signals that make proteins secretory. The SignalP web server has existed since 1995 and is now in its sixth major version. In this historical account, we (three authors who have taken part in the entire journey plus the first author of the latest version) describe the differences between the versions and discuss the various decisions taken along the way.
Asunto(s)
Internet , Señales de Clasificación de Proteína , Programas Informáticos , Biología Computacional/métodos , HumanosRESUMEN
Human growth hormone (hGH) is a four-helix bundle protein of considerable pharmacological interest. Recombinant hGH is produced in bacteria, yet little is known about its folding during expression in Escherichia coli. We have studied the cotranslational folding of hGH using force profile analysis (FPA), both during in vitro translation in the absence and presence of the chaperone trigger factor (TF), and when expressed in E. coli. We find that the main folding transition starts before hGH is completely released from the ribosome, and that it can interact with TF and possibly other chaperones.
Asunto(s)
Proteínas de Escherichia coli , Hormona de Crecimiento Humana , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Pliegue de Proteína , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/metabolismo , Biosíntesis de Proteínas , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
In recent years, it has become clear that many homo- and heterodimeric cytoplasmic proteins in both prokaryotic and eukaryotic cells start to dimerize cotranslationally (i.e., while at least one of the two chains is still attached to the ribosome). Whether this is also possible for integral membrane proteins is, however, unknown. Here, we apply force profile analysis (FPA)-a method where a translational arrest peptide (AP) engineered into the polypeptide chain is used to detect force generated on the nascent chain during membrane insertion-to demonstrate cotranslational interactions between a fully membrane-inserted monomer and a nascent, ribosome-tethered monomer of the Escherichia coli inner membrane protein EmrE. Similar cotranslational interactions are also seen when the two monomers are fused into a single polypeptide. Further, we uncover an apparent intrachain interaction between E14 in transmembrane helix 1 (TMH1) and S64 in TMH3 that forms at a precise nascent chain length during cotranslational membrane insertion of an EmrE monomer. Like soluble proteins, inner membrane proteins thus appear to be able to both start to fold and start to dimerize during the cotranslational membrane insertion process.
Asunto(s)
Antiportadores , Proteínas de Escherichia coli , Escherichia coli , Antiportadores/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Péptidos/metabolismo , Biosíntesis de Proteínas , Pliegue de ProteínaRESUMEN
During SecYEG-mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N-terminal end is ~ 45 residues away from the peptidyl transferase centre. However, we recently uncovered instances where the first contact is delayed by up to ~ 10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the Escherichia coli inner membrane protein BtuC: a positively charged loop and a re-entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re-entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion.
Asunto(s)
Proteínas de Escherichia coli , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Signal peptides (SPs) are short amino acid sequences that control protein secretion and translocation in all living organisms. SPs can be predicted from sequence data, but existing algorithms are unable to detect all known types of SPs. We introduce SignalP 6.0, a machine learning model that detects all five SP types and is applicable to metagenomic data.
Asunto(s)
Lenguaje , Señales de Clasificación de Proteína , Algoritmos , Secuencia de Aminoácidos , Señales de Clasificación de Proteína/genética , ProteínasRESUMEN
The ribosome stalling mechanism is a crucial biological process, yet its atomistic underpinning is still elusive. In this framework, the human XBP1u translational arrest peptide (AP) plays a central role in regulating the unfolded protein response (UPR) in eukaryotic cells. Here, we report multimicrosecond all-atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wild type AP and for four mutant variants of different arrest potencies. Enhanced sampling simulations allow investigating the AP release process of the different variants, shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP-ribosome interactions.
Asunto(s)
Péptidos , Ribosomas , Animales , Citosol , Humanos , Mamíferos , Simulación de Dinámica Molecular , Péptidos/química , Ribosomas/químicaRESUMEN
In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.
Asunto(s)
Proteínas de Escherichia coli/ultraestructura , Factores de Terminación de Péptidos/ultraestructura , Biosíntesis de Proteínas , Ribosomas/ultraestructura , Codón de Terminación/genética , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Conformación Proteica , Conformación Proteica en Hélice alfa , Ribosomas/genética , Triptófano/genéticaRESUMEN
This volume of the Annual Review of Biochemistry contains three reviews on membrane channel proteins: the first by Szczot et al., titled The Form and Function of PIEZO2; the second by Ruprecht & Kunji, titled Structural Mechanism of Transport of Mitochondrial Carriers; and the third by McIlwain et al., titled Membrane Exporters of Fluoride Ion. These reviews provide nice illustrations of just how far evolution has been able to play with the basic helix-bundle architecture of integral membrane proteins to produce membrane channels and transporters of widely different functions.
Asunto(s)
Canales Iónicos/química , Canales Iónicos/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Fluoruros/metabolismoRESUMEN
In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB's two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.
Asunto(s)
Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Canales de Translocación SEC/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/genética , Modelos Moleculares , Mutación , Biosíntesis de Proteínas , Conformación Proteica , Pliegue de Proteína , Serina Endopeptidasas/genéticaRESUMEN
Proteins commonly fold co-translationally at the ribosome, while the nascent chain emerges from the ribosomal exit tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not explain the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.
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Proteínas de Unión al ADN/química , Simulación de Dinámica Molecular , Biosíntesis de Proteínas , Pliegue de Proteína , Ribosomas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Proteínas de Unión al ADN/metabolismo , Dominios Proteicos , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the 'sliding' model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon.
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Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biosíntesis de Proteínas , Secuencias de Aminoácidos , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Dominios ProteicosRESUMEN
Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.
Asunto(s)
Fosfatasa Alcalina/biosíntesis , Proteínas de Escherichia coli/biosíntesis , Escherichia coli/enzimología , Periplasma/enzimología , Biosíntesis de Proteínas , Pliegue de Proteína , Fosfatasa Alcalina/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Periplasma/genéticaRESUMEN
Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using force profile analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other's folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in piconewtons. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of â¼15 pN during cotranslational folding.
Asunto(s)
Pliegue de Proteína , Espectrina/química , Escherichia coli , Simulación de Dinámica Molecular , Biosíntesis de Proteínas , Dominios Proteicos , Espectrina/genética , Espectrina/metabolismoRESUMEN
The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.
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Antígenos de Protozoos/química , Antígenos de Protozoos/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/fisiología , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Membrana Celular/química , Retículo Endoplásmico , Células HEK293 , Humanos , Conformación ProteicaRESUMEN
The TIM23 complex mediates membrane insertion of presequence-containing mitochondrial proteins via a stop-transfer mechanism. Stop-transfer signals consist of hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex. However, it remains elusive which features of stop-transfer signals are discriminated by Mgr2. To determine the effects of Mgr2 on the TIM23-mediated stop-transfer pathway, we measured membrane insertion of model transmembrane segments of varied hydrophobicity and flanking charges in Mgr2-deletion or -overexpression yeast strains. We found that upon deletion of Mgr2, the threshold hydrophobicity for membrane insertion, as well as the requirement for matrix-facing positive charges, is reduced. These results imply that the Mgr2-mediated gatekeeper function is important for controlling membrane sorting of marginal stop-transfer signals.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Mitocondrias/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In bioinformatics, machine learning methods have been used to predict features embedded in the sequences. In contrast to what is generally assumed, machine learning approaches can also provide new insights into the underlying biology. Here, we demonstrate this by presenting TargetP 2.0, a novel state-of-the-art method to identify N-terminal sorting signals, which direct proteins to the secretory pathway, mitochondria, and chloroplasts or other plastids. By examining the strongest signals from the attention layer in the network, we find that the second residue in the protein, that is, the one following the initial methionine, has a strong influence on the classification. We observe that two-thirds of chloroplast and thylakoid transit peptides have an alanine in position 2, compared with 20% in other plant proteins. We also note that in fungi and single-celled eukaryotes, less than 30% of the targeting peptides have an amino acid that allows the removal of the N-terminal methionine compared with 60% for the proteins without targeting peptide. The importance of this feature for predictions has not been highlighted before.
Asunto(s)
Biología Computacional/métodos , Péptidos/análisis , Péptidos/genética , Secuencia de Aminoácidos , Cloroplastos/genética , Cloroplastos/metabolismo , Aprendizaje Profundo , Hongos/genética , Hongos/metabolismo , Metionina/metabolismo , Señales de Clasificación de Proteína , Tilacoides/genética , Tilacoides/metabolismoRESUMEN
Helical membrane proteins are typically assumed to attain stable transmembrane topologies immediately upon co-translational membrane insertion. Here we show that unassembled monomers of the small multidrug resistance (SMR) family exist in a dynamic equilibrium where the N-terminal transmembrane helix flips in and out of the membrane, with rates that depend on dimerization and the polypeptide sequence. Thus, membrane topology can display rapid dynamics in vivo and can be regulated by post-translational assembly.
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Antiportadores/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/fisiología , Proteínas de la Membrana/química , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Antiportadores/genética , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Variación Genética , Proteínas de la Membrana/metabolismo , Plásmidos , Conformación ProteicaRESUMEN
XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.