RESUMO
The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Membranas Intracelulares/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios Proteicos , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
Intrinsically disordered proteins and regions (collectively, IDRs) are pervasive across proteomes in all kingdoms of life, help to shape biological functions and are involved in numerous diseases. IDRs populate a diverse set of transiently formed structures and defy conventional sequence-structure-function relationships1. Developments in protein science have made it possible to predict the three-dimensional structures of folded proteins at the proteome scale2. By contrast, there is a lack of knowledge about the conformational properties of IDRs, partly because the sequences of disordered proteins are poorly conserved and also because only a few of these proteins have been characterized experimentally. The inability to predict structural properties of IDRs across the proteome has limited our understanding of the functional roles of IDRs and how evolution shapes them. As a supplement to previous structural studies of individual IDRs3, we developed an efficient molecular model to generate conformational ensembles of IDRs and thereby to predict their conformational properties from sequences4,5. Here we use this model to simulate nearly all of the IDRs in the human proteome. Examining conformational ensembles of 28,058 IDRs, we show how chain compaction is correlated with cellular function and localization. We provide insights into how sequence features relate to chain compaction and, using a machine-learning model trained on our simulation data, show the conservation of conformational properties across orthologues. Our results recapitulate observations from previous studies of individual protein systems and exemplify how to link-at the proteome scale-conformational ensembles with cellular function and localization, amino acid sequence, evolutionary conservation and disease variants. Our freely available database of conformational properties will encourage further experimental investigation and enable the generation of hypotheses about the biological roles and evolution of IDRs.
Assuntos
Proteínas Intrinsicamente Desordenadas , Modelos Moleculares , Conformação Proteica , Proteoma , Humanos , Sequência de Aminoácidos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteoma/química , Proteoma/metabolismo , Relação Estrutura-Atividade , Evolução Molecular , Doença/genéticaRESUMO
In virus-host interactions, nucleic acid-directed first lines of defense that allow viral clearance without compromising growth are of paramount importance. Plants use the RNA interference pathway as a basal antiviral immune system, but additional RNA-based mechanisms of defense also exist. The infectivity of a plant positive-strand RNA virus, alfalfa mosaic virus (AMV), relies on the demethylation of viral RNA by the recruitment of the cellular N6-methyladenosine (m6 A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV infection remains unknown. Here, we show that inactivation of the Arabidopsis cytoplasmic YT521-B homology domain (YTH)-containing m6 A-binding proteins ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. We further show that the antiviral function of ECT2 is distinct from its previously demonstrated function in the promotion of primordial cell proliferation: an ect2 mutant carrying a small deletion in its intrinsically disordered region is partially compromised for antiviral defense but not for developmental functions. These results indicate that the m6 A-YTHDF axis constitutes a novel branch of basal antiviral immunity in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Vírus de RNA , Antivirais , Proteínas de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/metabolismo , RNA Viral/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na+ -dependent Cl- - and K+ -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+ , K+ , and 2 Cl- ) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- -ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl- -sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.
Assuntos
Potássio , Sódio , Membro 2 da Família 12 de Carreador de Soluto , Humanos , Microscopia Crioeletrônica , Potássio/metabolismo , Sódio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/químicaRESUMO
Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.
Assuntos
Compartimento Celular/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteostase/genética , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Humanos , Mutação , Neoplasias/patologia , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genéticaRESUMO
YT521-B homology (YTH) domain proteins act as readers of N6-methyladenosine (m6A) in mRNA. Members of the YTHDF clade determine properties of m6A-containing mRNAs in the cytoplasm. Vertebrates encode three YTHDF proteins whose possible functional specialization is debated. In land plants, the YTHDF clade has expanded from one member in basal lineages to eleven so-called EVOLUTIONARILY CONSERVED C-TERMINAL REGION1-11 (ECT1-11) proteins in Arabidopsis thaliana, named after the conserved YTH domain placed behind a long N-terminal intrinsically disordered region (IDR). ECT2, ECT3 and ECT4 show genetic redundancy in stimulation of primed stem cell division, but the origin and implications of YTHDF expansion in higher plants are unknown, as it is unclear whether it involves acquisition of fundamentally different molecular properties, in particular of their divergent IDRs. Here, we use functional complementation of ect2/ect3/ect4 mutants to test whether different YTHDF proteins can perform the same function when similarly expressed in leaf primordia. We show that stimulation of primordial cell division relies on an ancestral molecular function of the m6A-YTHDF axis in land plants that is present in bryophytes and is conserved over YTHDF diversification, as it appears in all major clades of YTHDF proteins in flowering plants. Importantly, although our results indicate that the YTH domains of all arabidopsis ECT proteins have m6A-binding capacity, lineage-specific neo-functionalization of ECT1, ECT9 and ECT11 happened after late duplication events, and involves altered properties of both the YTH domains, and, especially, of the IDRs. We also identify two biophysical properties recurrent in IDRs of YTHDF proteins able to complement ect2 ect3 ect4 mutants, a clear phase separation propensity and a charge distribution that creates electric dipoles. Human and fly YTHDFs do not have IDRs with this combination of properties and cannot replace ECT2/3/4 function in arabidopsis, perhaps suggesting different molecular activities of YTHDF proteins between major taxa.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Animais , Humanos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , RNA Mensageiro/metabolismo , Família Multigênica , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
Converting cryo-electron microscopy (cryo-EM) data into high-quality structural models is a challenging problem of outstanding importance. Current refinement methods often generate unbalanced models in which physico-chemical quality is sacrificed for excellent fit to the data. Furthermore, these techniques struggle to represent the conformational heterogeneity averaged out in low-resolution regions of density maps. Here we introduce EMMIVox, a Bayesian inference approach to determine single-structure models as well as structural ensembles from cryo-EM maps. EMMIVox automatically balances experimental information with accurate physico-chemical models of the system and the surrounding environment, including waters, lipids, and ions. Explicit treatment of data correlation and noise as well as inference of accurate B-factors enable determination of structural models and ensembles with both excellent fit to the data and high stereochemical quality, thus outperforming state-of-the-art refinement techniques. EMMIVox represents a flexible approach to determine high-quality structural models that will contribute to advancing our understanding of the molecular mechanisms underlying biological functions.
Assuntos
Teorema de Bayes , Microscopia Crioeletrônica , Modelos Moleculares , Microscopia Crioeletrônica/métodos , Biologia Computacional/métodos , Conformação Proteica , Algoritmos , Proteínas/química , Proteínas/ultraestruturaRESUMO
Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Sequência Conservada , Cristalografia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Genótipo , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Ressonância Magnética Nuclear Biomolecular , Fenótipo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Relação Estrutura-AtividadeRESUMO
Parkinson's disease (PD) is a common and incurable neurodegenerative disorder that arises from the loss of dopaminergic neurons in the substantia nigra and is mainly characterized by progressive loss of motor function. Monogenic familial PD is associated with highly penetrant variants in specific genes, notably the PRKN gene, where homozygous or compound heterozygous loss-of-function variants predominate. PRKN encodes Parkin, an E3 ubiquitin-protein ligase important for protein ubiquitination and mitophagy of damaged mitochondria. Accordingly, Parkin plays a central role in mitochondrial quality control but is itself also subject to a strict protein quality control system that rapidly eliminates certain disease-linked Parkin variants. Here, we summarize the cellular and molecular functions of Parkin, highlighting the various mechanisms by which PRKN gene variants result in loss-of-function. We emphasize the importance of high-throughput assays and computational tools for the clinical classification of PRKN gene variants and how detailed insights into the pathogenic mechanisms of PRKN gene variants may impact the development of personalized therapeutics.
Assuntos
Doença de Parkinson , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Doença de Parkinson/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/patologia , Ubiquitinação/genética , Mitofagia/genética , AnimaisRESUMO
PGM1-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in S. cerevisiae and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.
Assuntos
Fosfoglucomutase , Saccharomyces cerevisiae , Humanos , Fosfoglucomutase/metabolismo , Fosfoglucomutase/genética , Fosfoglucomutase/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteólise , Mutação de Sentido Incorreto , Ubiquitinação , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Estabilidade Proteica , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genéticaRESUMO
A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering.
Assuntos
Proteínas Fúngicas , Oxigenases de Função Mista , Modelos Moleculares , Dobramento de Proteína , Domínio Catalítico , Celulose/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Serina , Estabilidade Proteica , Ativação Enzimática , Simulação de Acoplamento Molecular , Streptomyces/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Estrutura Terciária de ProteínaRESUMO
In terms of its relative frequency, lysine is a common amino acid in the human proteome. However, by bioinformatics we find hundreds of proteins that contain long and evolutionarily conserved stretches completely devoid of lysine residues. These so-called lysine deserts show a high prevalence in intrinsically disordered proteins with known or predicted functions within the ubiquitin-proteasome system (UPS), including many E3 ubiquitin-protein ligases and UBL domain proteasome substrate shuttles, such as BAG6, RAD23A, UBQLN1 and UBQLN2. We show that introduction of lysine residues into the deserts leads to a striking increase in ubiquitylation of some of these proteins. In case of BAG6, we show that ubiquitylation is catalyzed by the E3 RNF126, while RAD23A is ubiquitylated by E6AP. Despite the elevated ubiquitylation, mutant RAD23A appears stable, but displays a partial loss of function phenotype in fission yeast. In case of UBQLN1 and BAG6, introducing lysine leads to a reduced abundance due to proteasomal degradation of the proteins. For UBQLN1 we show that arginine residues within the lysine depleted region are critical for its ability to form cytosolic speckles/inclusions. We propose that selective pressure to avoid lysine residues may be a common evolutionary mechanism to prevent unwarranted ubiquitylation and/or perhaps other lysine post-translational modifications. This may be particularly relevant for UPS components as they closely and frequently encounter the ubiquitylation machinery and are thus more susceptible to nonspecific ubiquitylation.
Assuntos
Complexo de Endopeptidases do Proteassoma , Schizosaccharomyces , Humanos , Ubiquitina , Lisina , Citoplasma , Ubiquitinação , Schizosaccharomyces/genética , Chaperonas Moleculares , Proteínas Relacionadas à Autofagia , Proteínas Adaptadoras de Transdução de Sinal , Ubiquitina-Proteína LigasesRESUMO
Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteólise , Dobramento de Proteína , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Many intrinsically disordered proteins (IDPs) may undergo liquid-liquid phase separation (LLPS) and participate in the formation of membraneless organelles in the cell, thereby contributing to the regulation and compartmentalization of intracellular biochemical reactions. The phase behavior of IDPs is sequence dependent, and its investigation through molecular simulations requires protein models that combine computational efficiency with an accurate description of intramolecular and intermolecular interactions. We developed a general coarse-grained model of IDPs, with residue-level detail, based on an extensive set of experimental data on single-chain properties. Ensemble-averaged experimental observables are predicted from molecular simulations, and a data-driven parameter-learning procedure is used to identify the residue-specific model parameters that minimize the discrepancy between predictions and experiments. The model accurately reproduces the experimentally observed conformational propensities of a set of IDPs. Through two-body as well as large-scale molecular simulations, we show that the optimization of the intramolecular interactions results in improved predictions of protein self-association and LLPS.
Assuntos
Condensados Biomoleculares/química , Condensados Biomoleculares/fisiologia , Proteínas Intrinsicamente Desordenadas/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Teóricos , Organelas/química , Organelas/fisiologia , Mapas de Interação de ProteínasRESUMO
Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.
Assuntos
Amidoidrolases/genética , Doença de Canavan/genética , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP70/genética , Substituição de Aminoácidos/genética , Doença de Canavan/patologia , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The rapid decrease in DNA sequencing cost is revolutionizing medicine and science. In medicine, genome sequencing has revealed millions of missense variants that change protein sequences, yet we only understand the molecular and phenotypic consequences of a small fraction. Within protein science, high-throughput deep mutational scanning experiments enable us to probe thousands of variants in a single, multiplexed experiment. We review efforts that bring together these topics via experimental and computational approaches to determine the consequences of missense variants in proteins. We focus on the role of changes in protein stability as a driver for disease, and how experiments, biophysical models, and computation are providing a framework for understanding and predicting how changes in protein sequence affect cellular protein stability.
Assuntos
Doença/genética , Variação Genética/genética , Modelos Genéticos , Proteínas/genética , Biologia Computacional , Humanos , Mutação de Sentido Incorreto , Estabilidade ProteicaRESUMO
Next-generation sequencing of human genomes reveals millions of missense variants, some of which may lead to loss of protein function and ultimately disease. Here, we investigate missense variants in membrane proteins-key drivers in cell signaling and recognition. We find enrichment of pathogenic variants in the transmembrane region across 19,000 functionally classified variants in human membrane proteins. To accurately predict variant consequences, one fundamentally needs to understand the underlying molecular processes. A key mechanism underlying pathogenicity in missense variants of soluble proteins has been shown to be loss of stability. Membrane proteins, however, are widely understudied. Here, we interpret variant effects on a larger scale by performing structure-based estimations of changes in thermodynamic stability using a membrane-specific energy function and analyses of sequence conservation during evolution of 15 transmembrane proteins. We find evidence for loss of stability being the cause of pathogenicity in more than half of the pathogenic variants, indicating that this is a driving factor also in membrane-protein-associated diseases. Our findings show how computational tools aid in gaining mechanistic insights into variant consequences for membrane proteins. To enable broader analyses of disease-related and population variants, we include variant mappings for the entire human proteome.
Assuntos
Proteínas de Membrana , Transdução de Sinais , Humanos , Proteínas de Membrana/genética , Mutação de Sentido IncorretoRESUMO
Diffusion measurements by pulsed-field gradient NMR and fluorescence correlation spectroscopy can be used to probe the hydrodynamic radius of proteins, which contains information about the overall dimension of a protein in solution. The comparison of this value with structural models of intrinsically disordered proteins is nonetheless impaired by the uncertainty of the accuracy of the methods for computing the hydrodynamic radius from atomic coordinates. To tackle this issue, we here build conformational ensembles of 11 intrinsically disordered proteins that we ensure are in agreement with measurements of compaction by small-angle x-ray scattering. We then use these ensembles to identify the forward model that more closely fits the radii derived from pulsed-field gradient NMR diffusion experiments. Of the models we examined, we find that the Kirkwood-Riseman equation provides the best description of the hydrodynamic radius probed by pulsed-field gradient NMR experiments. While some minor discrepancies remain, our results enable better use of measurements of the hydrodynamic radius in integrative modeling and for force field benchmarking and parameterization.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Rádio (Anatomia)/metabolismo , Hidrodinâmica , Conformação Proteica , Espectrometria de Fluorescência , Espalhamento a Baixo ÂnguloRESUMO
Catechol-O-methyltransferase (COMT) is a key enzyme in the metabolism of catecholamines. Substrates of the enzyme include neurotransmitters such as dopamine and epinephrine, and therefore, COMT plays a central role in neurobiology. Since COMT also metabolizes catecholamine drugs such as L-DOPA, variation in COMT activity could affect pharmacokinetics and drug availability. Certain COMT missense variants have been shown to display decreased enzymatic activity. Additionally, studies have shown that such missense variants may lead to loss of function induced by impaired structural stability, which results in activation of the protein quality control system and degradation by the ubiquitin-proteasome system. Here, we demonstrate that two rare missense variants of COMT are ubiquitylated and targeted for proteasomal degradation as a result of structural destabilization and misfolding. This results in strongly reduced intracellular steady-state levels of the enzyme, which for the L135P variant is rescued upon binding to the COMT inhibitors entacapone and tolcapone. Our results reveal that the degradation is independent of the COMT isoform as both soluble (S-COMT) and ER membrane-bound (MB-COMT) variants are degraded. In silico structural stability predictions identify regions within the protein that are critical for stability overlapping with evolutionarily conserved residues, pointing toward other variants that are likely destabilized and degraded.
Assuntos
Catecol O-Metiltransferase , Complexo de Endopeptidases do Proteassoma , Catecol O-Metiltransferase/genética , Complexo de Endopeptidases do Proteassoma/genética , Tolcapona , Inibidores de Catecol O-Metiltransferase/farmacologia , Levodopa , Catecolaminas/metabolismoRESUMO
Both experimental and theoretical structure determinations of RNAs have remained challenging due to the intrinsic dynamics of RNAs. We report here an integrated nuclear magnetic resonance/molecular dynamics (NMR/MD) structure determination approach to describe the dynamic structure of the CUUG tetraloop. We show that the tetraloop undergoes substantial dynamics, leading to averaging of the experimental data. These dynamics are particularly linked to the temperature-dependent presence of a hydrogen bond within the tetraloop. Interpreting the NMR data by a single structure represents the low-temperature structure well but fails to capture all conformational states occurring at a higher temperature. We integrate MD simulations, starting from structures of CUUG tetraloops within the Protein Data Bank, with an extensive set of NMR data, and provide a structural ensemble that describes the dynamic nature of the tetraloop and the experimental NMR data well. We thus show that one of the most stable and frequently found RNA tetraloops displays substantial dynamics, warranting such an integrated structural approach.