RESUMEN
Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins1-3. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour3,4. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors5-9. Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P2, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.
Asunto(s)
Microscopía por Crioelectrón , Microdominios de Membrana , Saccharomyces cerevisiae , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Microdominios de Membrana/ultraestructura , Modelos Moleculares , Simulación de Dinámica Molecular , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/ultraestructura , Dominios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Esteroles/química , Esteroles/metabolismo , Estrés MecánicoRESUMEN
Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.
Asunto(s)
Síndromes de Inmunodeficiencia , Linfopenia , Lactante , Humanos , Animales , Ratones , Antígenos CD28 , Linfocitos T CD4-Positivos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Receptores de Antígenos de Linfocitos T/genética , Inflamación/genética , Linfopenia/genéticaRESUMEN
The human leukocyte antigen (HLA)-B*27 family of alleles is strongly associated with ankylosing spondylitis (AS), a chronic inflammatory disorder affecting the axial and peripheral joints, yet some HLA-B*27 variants not associated with AS have been shown. Since no major differences in the ligandome of associated compared to not-associated alleles have emerged, a plausible hypothesis is that the quantity rather than the quality of the presented epitopes makes the difference. In addition, the Endoplasmic Reticulum AminoPeptidases (ERAPs) 1 and 2, playing a crucial role in shaping the HLA class I epitopes, act as strong AS susceptibility factors, suggesting that an altered peptidome might be responsible for the activation of pathogenic CD8+ T cells. In this context, we have previously singled out a B*27:05-restricted CD8+ T cell response against pEBNA3A (RPPIFIRRL), an EBV peptide lacking the B*27 classic binding motif. Here, we show that a specific ERAP1/2 haplotype negatively correlates with such response in B*27:05 subjects. Moreover, we prove that the B*27:05 allele successfully presents peptides with the same suboptimal N-terminal RP motif, including the self-peptide, pDYNEIN (RPPIFGDFL). Overall, this study underscores the cooperation between the HLA-B*27 and ERAP1/2 allelic variants in defining CD8+ T cell reactivity to suboptimal viral and self-B*27 peptides and prompts further investigation of the B*27:05 peptidome composition.
Asunto(s)
Genes MHC Clase I , Espondilitis Anquilosante , Humanos , Haplotipos , Antígenos HLA-B/genética , Linfocitos T CD8-positivos , Epítopos , Espondilitis Anquilosante/genética , Aminopeptidasas/genética , Antígenos de Histocompatibilidad Menor/genéticaRESUMEN
Cell growth is regulated by the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here we show that mTORC1 senses the capacity of a cell to synthesise fatty acids by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When fatty acid synthase (FASN) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with acetyl-CoA carboxylase 1 (ACC1) and FASN. Our findings represent a conserved and unique homeostatic mechanism whereby impaired fatty acid biogenesis leads to reduced mTORC1 activity to coordinately link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the existence of a physiological metabolite that directly inhibits the activity of a signalling kinase in mammalian cells by competing with ATP for binding.
Asunto(s)
Acetil-CoA Carboxilasa , Malonil Coenzima A , Animales , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Malonil Coenzima A/metabolismo , Serina-Treonina Quinasas TOR/genética , Ácidos Grasos/metabolismo , Mamíferos/metabolismo , Adenosina TrifosfatoRESUMEN
The machinery involved in cytotoxic T-cell activation requires three main characters: the major histocompatibility complex class I (MHC I) bound to the peptide (p), the T-cell receptor (TCR), and the CD3 complex, a multidimer interfaced with the intracellular side. The pMHC:TCR interaction has been largely studied by means of both experimental and computational models, giving a contribution in understanding the complexity of the TCR triggering. Nevertheless, a detailed study of the structural and dynamical characterization of the full complex (pMHC:TCR:CD3 complex) is still missing due to a lack of structural information of the CD3-chains arrangement around the TCR. Very recently, the determination of the TCR:CD3 complex structure by means of Cryo-EM technique has given a chance to build the entire system essential in the activation of T-cells, a fundamental mechanism in the adaptive immune response. Here, we present the first complete model of the pMHC interacting with the TCR:CD3 complex, built in a lipid environment. To describe the conformational behavior associated with the unbound and the bound states, all-atom Molecular Dynamics simulations were performed for the TCR:CD3 complex and for two pMHC:TCR:CD3 complex systems, bound to two different peptides. Our data point out that a conformational change affecting the TCR Constant ß (Cß) region occurs after the binding to the pMHC, revealing a key role of this region in the propagation of the signal. Moreover, we found that TCR reduces the flexibility of the MHC I binding groove, confirming our previous results.
Asunto(s)
Complejo Mayor de Histocompatibilidad , Complejo Receptor-CD3 del Antígeno de Linfocito T , Complejo CD3/metabolismo , Péptidos/metabolismo , Unión Proteica , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/químicaRESUMEN
Human hemoglobin (HbA) is one of the prototypal systems used to investigate structure-function relationships in proteins. Indeed, HbA has been used to develop the basic concepts of protein allostery, although the atomic-level mechanism underlying the HbA functionality is still highly debated. This is due to the fact that most of the three-dimensional structural information collected over the decades refers to the endpoints of HbA functional transition with little data available for the intermediate states. Here, we report molecular dynamics (MD) simulations by focusing on the relevance of the intermediate states of the protein functional transition unraveled by the crystallographic studies carried out on vertebrate Hbs. Fully atomistic simulations of the HbA T-state indicate that the protein undergoes a spontaneous transition toward the R-state. The inspection of the trajectory structures indicates that the protein significantly populates the intermediate HL-(C) state previously unraveled by crystallography. In the structural transition, it also assumes the intermediate states crystallographically detected in Antarctic fish Hbs. This finding suggests that HbA and Antarctic fish Hbs, in addition to the endpoints of the transitions, also share a similar deoxygenation pathway despite a distace of hundreds of millions of years in the evolution scale. Finally, using the essential dynamic sampling methodology, we gained some insights into the reverse R to T transition that is not spontaneously observed in classic MD simulations.
Asunto(s)
Hemoglobinas , Simulación de Dinámica Molecular , Animales , Cristalografía , Humanos , Estructura Cuaternaria de ProteínaRESUMEN
The major defense system against microbial pathogens in vertebrates is the adaptive immune response and represents an effective mechanism in cancer surveillance. T cells represent an essential component of this complex system. They can recognize myriads of antigens as short peptides (p) originated from the intracellular degradation of foreign proteins presented by major histocompatibility complex (MHC) proteins. The clonotypic T-cell antigen receptor (TCR) is specialized in recognizing pMHC and triggering T cells immune response. It is still unclear how TCR engagement to pMHC is translated into the intracellular signal that initiates T-cell immune response. Some work has suggested the possibility that pMHC binding induces in the TCR conformational changes transmitted to its companion CD3 subunits that govern signaling. The conformational changes would promote phosphorylation of the CD3 complex ζ chain that initiates signal propagation intracellularly. Here, we used all-atom molecular dynamics simulations (MDs) of 500 ns to analyze the conformational behavior of three TCRs (1G4, ILA1 and ILA1α1ß1) interacting with the same MHC class I (HLA-A*02:01) bound to different peptides, and modelled in the presence of a lipid bilayer. Our data suggest a correlation between the conformations explored by the ß-chain constant regions and the T-cell response experimentally determined. In particular, independently by the TCR type involved in the interaction, the TCR activation seems to be linked to a specific zone of the conformational space explored by the ß-chain constant region. Moreover, TCR ligation restricts the conformational space the MHC class I groove.
Asunto(s)
Complejo Mayor de Histocompatibilidad/genética , Simulación de Dinámica Molecular/normas , Receptores de Antígenos de Linfocitos T/metabolismo , HumanosRESUMEN
In this work, we describe the application of the Zernike formalism to quantitatively characterize the binding pockets of two sets of biologically relevant systems. Such an approach, when applied to molecular dynamics trajectories, is able to pinpoint the subtle differences between very similar molecular regions and their impact on the local propensity to ligand binding, allowing us to quantify such differences. The statistical robustness of our procedure suggests that it is very suitable to describe protein binding sites and protein-ligand interactions within a rigorous and well-defined framework.
Asunto(s)
Proteínas , Sitios de Unión , Ligandos , Unión Proteica , Conformación Proteica , Proteínas/metabolismoRESUMEN
Using a combined approach based on MS, enzyme digestion and advanced MD studies we have determined the sequential order of formation of the three disulfide bridges of the Cripto-1 CFC domain. The domain has a rare pattern of bridges and is involved in the recognition of several receptors. The bridge formation order is C1-C4, C3-C5, C2-C6, however formation of C1-C4 plays no roles for the formation of the others. Folding is driven by formation of the C3-C5 bridge and is supported by residues lying within the segment delimited by these cysteines. We indeed observe that variants CFC-W123A and CFC-ΔC1/C4, where C1 and C4 are replaced by serines, are able to refold in the same time window as the wild type, while CFC-K132A and CFC-W134A are not. A variant where cysteines of the second and third bridge are mutated to serine, convert slowly to the monocyclic molecule. Data altogether support a mechanism whereby the Cripto-1 CFC domain refolds by virtue of long-range intramolecular interactions that involve residues close to cysteines of the second and third bridge. These findings are supported by the in silico study that shows how distant parts of the molecules come into contact on a long time scale.
Asunto(s)
Proteínas Ligadas a GPI/química , Péptidos y Proteínas de Señalización Intercelular/química , Proteínas de Neoplasias/química , Replegamiento Proteico , Secuencia de Aminoácidos , Disulfuros/química , Proteínas Ligadas a GPI/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cinética , Simulación de Dinámica Molecular , Proteínas de Neoplasias/metabolismo , Oxidación-Reducción , Fragmentos de Péptidos/química , Dominios ProteicosRESUMEN
The human leukocyte antigen HLA-B27 is a strong risk factor for Ankylosing Spondylitis (AS), an immune-mediated disorder affecting axial skeleton and sacroiliac joints. Additionally, evidence exists sustaining a strong protective role for HLA-B27 in viral infections. These two aspects could stem from common molecular mechanisms. Recently, we have found that the HLA-B*2705 presents an EBV epitope (pEBNA3A-RPPIFIRRL), lacking the canonical B27 binding motif but known as immunodominant in the HLA-B7 context of presentation. Notably, 69% of B*2705 carriers, mostly patients with AS, possess B*2705-restricted, pEBNA3A-specific CD8+ T cells. Contrarily, the non-AS-associated B*2709 allele, distinguished from the B*2705 by the single His116Asp polymorphism, is unable to display this peptide and, accordingly, B*2709 healthy subjects do not unleash specific T cell responses. Herein, we investigated whether the reactivity towards pEBNA3A could be a side effect of the recognition of the natural longer peptide (pKEBNA3A) having the classical B27 consensus (KRPPIFIRRL). The stimulation of PBMC from B*2705 positive patients with AS in parallel with both pEBNA3A and pKEBNA3A did not allow to reach an unambiguous conclusion since the differences in the magnitude of the response measured as percentage of IFNγ-producing CD8+ T cells were not statistically significant. Interestingly, computational analysis suggested a structural shift of pEBNA3A as well as of pKEBNA3A into the B27 grooves, leaving the A pocket partially unfilled. To our knowledge this is the first report of a viral peptide: HLA-B27 complex recognized by TCRs in spite of a partially empty groove. This implies a rethinking of the actual B27 immunopeptidome crucial for viral immune-surveillance and autoimmunity.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Epítopos/inmunología , Antígeno HLA-B27/genética , Herpesvirus Humano 4/metabolismo , Espondilitis Anquilosante/diagnóstico , Alelos , Secuencia de Aminoácidos , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Epítopos/química , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos , Plásmidos/genética , Plásmidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Espondilitis Anquilosante/inmunología , Espondilitis Anquilosante/metabolismo , Espondilitis Anquilosante/patologíaRESUMEN
Here we report the description of the conformational pathways connecting the Lck active and inactive states by means of all-atoms molecular dynamics simulations coupled to an enhancing sampling methodology. By such an approach, we describe the major structural determinants characterizing these large conformational transitions and compare such pathways to those obtained for a similar kinase, i.e. c-Src. Our results show that both the activation and deactivation processes could follow distinct pathways, differentiated by the order by which the A-loop and the C-helix regions rearrange.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Activación Enzimática , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Conformación Proteica en Hélice alfa , TermodinámicaRESUMEN
In this article, we investigated the structural and dynamical evolutionary behaviour of a set of ten thioredoxin proteins as formed by three extant forms and seven resurrected ones in laboratory. Starting from the crystallographic structures, we performed all-atom molecular dynamics simulations and compare the trajectories in terms of structural and dynamical properties. Interestingly, the structural properties related to the protein density (i.e. the number of residues divided by the excluded molecular volume) well describe the protein evolutionary behaviour. Our results also suggest that the changes in sequence as occurred during the evolution have affected the protein essential motions, allowing us to discriminate between ancient and extant proteins in terms of their dynamical behaviour. Such results are yet more evident when the bacterial, archaeal and eukaryotic thioredoxins are separately analysed.
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Archaea/genética , Bacterias/genética , Evolución Molecular , Tiorredoxinas/genética , Regulación de la Expresión Génica , Humanos , Simulación de Dinámica Molecular , Análisis de Componente Principal , Conformación Proteica , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMEN
The theoretical computational modeling of large conformational transitions occurring in biomolecules still represents a challenge. Here, we present an accurate "in silico" description of the activation and deactivation mechanisms of human c-Src kinases, a fundamental process regulating several crucial cell functions. Our results clearly show that by applying an efficient and automated algorithm able to drive the molecular dynamics (MD) sampling along the pathway between the two c-Src conformational states-the active state and the inactive state-it is possible to accurately describe, at reduced computational costs, the molecular mechanism underlying these large conformational rearrangements. This procedure, combining the MD simulations with the sampling along the well-defined principal motions connecting the two conformational states, allows to provide a description well beyond the present computational limits, and it is easily applicable to different systems where the structures of both the initial and final states are known.
Asunto(s)
Familia-src Quinasas/química , Proteína Tirosina Quinasa CSK , Humanos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
I-DmoI, from the hyperthermophilic archaeon Desulfurococcus mobilis, belongs to the LAGLIDADG homing endonuclease protein family. Its members are highly specific enzymes capable of recognizing long DNA target sequences, thus providing potential tools for genome manipulation. Working towards this particular application, many efforts have been made to generate mesophilic variants of I-DmoI that function at lower temperatures than the wild-type. Here, we report a structural and computational analysis of two I-DmoI mesophilic mutants. Despite very limited structural variations between the crystal structures of these variants and the wild-type, a different dynamical behaviour near the cleavage sites is observed. In particular, both the dynamics of the water molecules and the protein perturbation effect on the cleavage site correlate well with the changes observed in the experimental enzymatic activity.