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Hypertension affects one billion people worldwide and is the most common risk factor for cardiovascular disease, yet a comprehensive picture of its underlying genetic factors is incomplete. Amongst regulators of blood pressure is the renal outer medullary potassium (ROMK) channel. While select ROMK mutants are prone to premature degradation and lead to disease, heterozygous carriers of some of these same alleles are protected from hypertension. Therefore, we hypothesized that gain-of-function (GoF) ROMK variants which increase potassium flux may predispose people to hypertension. To begin to test this hypothesis, we employed genetic screens and a candidate-based approach to identify six GoF variants in yeast. Subsequent functional assays in higher cells revealed two variant classes. The first group exhibited greater stability in the endoplasmic reticulum, enhanced channel assembly, and/or increased protein at the cell surface. The second group of variants resided in the PIP2-binding pocket, and computational modeling coupled with patch-clamp studies demonstrated lower free energy for channel opening and slowed current rundown, consistent with an acquired PIP2-activated state. Together, these findings advance our understanding of ROMK structure-function, suggest the existence of hyperactive ROMK alleles in humans, and establish a system to facilitate the development of ROMK-targeted antihypertensives.
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Canales de Potasio de Rectificación Interna , Humanos , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Rectificación Interna/genética , Mutación con Ganancia de Función , Potasio/metabolismo , Hipertensión/genética , Hipertensión/metabolismo , Riñón/metabolismo , Mutación/genética , Células HEK293 , Retículo Endoplásmico/metabolismo , Transporte Iónico , AlelosRESUMEN
PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.
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Simulación de Dinámica Molecular , Pinzas Ópticas , Mutación Puntual , Conformación Proteica , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/metabolismo , HumanosRESUMEN
During B cell development, cells progress through multiple developmental stages with the pro-B cell stage defining commitment to the B cell lineage. YY1 is a ubiquitous transcription factor that is capable of both activation and repression functions. We find here that knockout of YY1 at the pro-B cell stage eliminates B lineage commitment. YY1 knockout pro-B cells can generate T lineage cells in vitro using the OP9- DL4 feeder system, as well as in vivo after injection into sub-lethally irradiated Rag1 -/- mice. These T lineage-like cells lose their B lineage transcript profile and gain a T cell lineage profile. Single cell-RNA-seq experiments showed that as YY1 knockout pro-B cells transition into T lineage cells, various cell clusters adopt transcript profiles representing a multiplicity of hematopoietic lineages indicating unusual lineage plasticity. Given the ubiquitous nature of YY1 and its dual activation and repression functions, YY1 likely regulates commitment in multiple cell lineages.
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Characterization of the spatiotemporal properties of the chromatin is essential to gaining insights into the physical bases of gene co-expression, transcriptional regulation and epigenetic modifications. The Gaussian network model (GNM) has proven in recent work to serve as a useful tool for modeling chromatin structural dynamics, using as input high-throughput chromosome conformation capture data. We focus here on the exploration of the collective dynamics of chromosomal structures at hierarchical levels of resolution, from single gene loci to topologically associating domains or entire chromosomes. The GNM permits us to identify long-range interactions between gene loci, shedding light on the role of cross-correlations between distal regions of the chromosomes in regulating gene expression. Notably, GNM analysis performed across diverse cell lines highlights the conservation of the global/cooperative movements of the chromatin across different types of cells. Variations driven by localized couplings between genomic loci, on the other hand, underlie cell differentiation, underscoring the significance of the four-dimensional properties of the genome in defining cellular identity. Finally, we demonstrate the close relation between the cell type-dependent mobility profiles of gene loci and their gene expression patterns, providing a clear demonstration of the role of chromosomal 4D features in defining cell-specific differential expression of genes.
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Despite widespread utilization of immunotherapy, treating immune-cold tumors remains a challenge. Multiomic analyses and experimental validation identified the OTUD4/CD73 proteolytic axis as a promising target in treating immune-suppressive triple negative breast cancer (TNBC). Mechanistically, deubiquitylation of CD73 by OTUD4 counteracted its ubiquitylation by TRIM21, resulting in CD73 stabilization inhibiting tumor immune responses. We further demonstrated the importance of TGF-ß signaling for orchestrating the OTUD4/CD73 proteolytic axis within tumor cells. Spatial transcriptomics profiling discovered spatially resolved features of interacting malignant and immune cells pertaining to expression levels of OTUD4 and CD73. In addition, ST80, a newly developed inhibitor, specifically disrupted proteolytic interaction between CD73 and OTUD4, leading to reinvigoration of cytotoxic CD8+ T cell activities. In preclinical models of TNBC, ST80 treatment sensitized refractory tumors to anti-PD-L1 therapy. Collectively, our findings uncover what we believe to be a novel strategy for targeting the immunosuppressive OTUD4/CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80.
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5'-Nucleotidasa , Proteolisis , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/inmunología , 5'-Nucleotidasa/antagonistas & inhibidores , Línea Celular Tumoral , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Ubiquitinación , Proteasas Ubiquitina-EspecíficasRESUMEN
The architectures of tandem-repeat proteins are distinct from those of globular proteins. Individual modules, each comprising small structural motifs of 20-40 residues, are arrayed in a quasi one-dimensional fashion to form striking, elongated, horseshoe-like, and superhelical architectures, stabilized solely by short-range interaction. The spring-like shapes of repeat arrays point to elastic modes of action, and these proteins function as adapter molecules or 'hubs,' propagating signals within multi-subunit assemblies in diverse biological contexts. This flexibility is apparent in the dramatic variability observed in the structures of tandem-repeat proteins in different complexes. Here, using computational analysis, we demonstrate the striking ability of just one or a few global motions to recapitulate these structures. These findings show how the mechanics of repeat arrays are robustly enabled by their unique architecture. Thus, the repeating architecture has been optimized by evolution to favor functional modes of motions. The global motions enabling functional transitions can be fully visualized at http://bahargroup.org/tr_web.
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Proteínas , Programas Informáticos , Conformación Proteica , Proteínas/química , Movimiento (Física)RESUMEN
PR65 is the HEAT-repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem-repeat protein, forming a spring-like architecture. PR65 conformational mechanics play a crucial role in PP2A function by opening/closing the substrate-binding/catalysis interface. Using in-silico saturation mutagenesis we identified "hinge" residues of PR65, whose substitutions are predicted to restrict its conformational adaptability and thereby disrupt PP2A function. Molecular simulations revealed that a subset of hinge mutations stabilized the extended/open conformation, whereas another had the opposite effect. By trapping in nanoaperture optical tweezer, we characterized PR65 motion and showed that the former mutants exhibited higher corner frequencies and lower translational scattering, indicating a shift towards extended conformations, whereas the latter showed the opposite behavior. Thus, experiments confirm the conformations predicted computationally. The study highlights the utility of nanoaperture-based tweezers for exploring structure and dynamics, and the power of integrating this single-molecule method with in silico approaches.
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The insertion or deletion (indel) of amino acids has a variety of effects on protein function, ranging from disease-forming changes to gaining new functions. Despite their importance, indels have not been systematically characterized towards protein engineering or modification goals. In the present work, we focus on deletions composed of multiple contiguous amino acids (mAA-dels) and their effects on the protein (mutant) folding ability. Our analysis reveals that the mutant retains the native fold when the mAA-del obeys well-defined structural dynamics properties: localization in intrinsically flexible regions, showing low resistance to mechanical stress, and separation from allosteric signaling paths. Motivated by the possibility of distinguishing the features that underlie the adaptability of proteins to mAA-dels, and by the rapid evaluation of these features using elastic network models, we developed a positive-unlabeled learning-based classifier that can be adopted for protein design purposes. Trained on a consolidated set of features, including those reflecting the intrinsic dynamics of the regions where the mAA-dels occur, the new classifier yields a high recall of 84.3% for identifying mAA-dels that are stably tolerated by the protein. The comparative examination of the relative contribution of different features to the prediction reveals the dominant role of structural dynamics in enabling the adaptation of the mutant to mAA-del without disrupting the native fold.
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Aminoácidos , Proteínas , Aminoácidos/genética , Proteínas/química , Mutación INDEL , Ingeniería de ProteínasRESUMEN
PR65, a horseshoe-shaped scaffold composed of 15 HEAT (observed in Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) repeats, forms, together with catalytic and regulatory subunits, the heterotrimeric protein phosphatase PP2A. We examined the role of PR65 in enabling PP2A enzymatic activity with computations at various levels of complexity, including hybrid approaches that combine full-atomic and elastic network models. Our study points to the high flexibility of this scaffold allowing for end-to-end distance fluctuations of 40-50 Å between compact and extended conformations. Notably, the intrinsic dynamics of PR65 facilitates complexation with the catalytic subunit and is retained in the PP2A complex enabling PR65 to engage the two domains of the catalytic subunit and provide the mechanical framework for enzymatic activity, with support from the regulatory subunit. In particular, the intra-repeat coils at the C-terminal arm play an important role in allosterically mediating the collective dynamics of PP2A, pointing to target sites for modulating PR65 function.
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Proteína Fosfatasa 2 , Proteína Fosfatasa 2/genética , Regulación Alostérica , Unión Proteica , Dominio CatalíticoRESUMEN
Proteins sample an ensemble of conformers under physiological conditions, having access to a spectrum of modes of motions, also called intrinsic dynamics. These motions ensure the adaptation to various interactions in the cell, and largely assist in, if not determine, viable mechanisms of biological function. In recent years, machine learning frameworks have proven uniquely useful in structural biology, and recent studies further provide evidence to the utility and/or necessity of considering intrinsic dynamics for increasing their predictive ability. Efficient quantification of dynamics-based attributes by recently developed physics-based theories and models such as elastic network models provides a unique opportunity to generate data on dynamics for training ML models towards inferring mechanisms of protein function, assessing pathogenicity, or estimating binding affinities.
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Aprendizaje Automático , Proteínas , Proteínas/químicaRESUMEN
De-novo protein design explores the untapped sequence space that is otherwise less discovered during the evolutionary process. This necessitates an efficient sequence space search engine for effective convergence in computational protein design. We propose a greedy simulated annealing-based Monte-Carlo parallel search algorithm for better sequence-structure compatibility probing in protein design. The guidance provided by the evolutionary profile, the greedy approach, and the cooling schedule adopted in the Monte Carlo simulation ensures sufficient exploration and exploitation of the search space leading to faster convergence. On evaluating the proposed algorithm, we find that a dataset of 76 target scaffolds report an average root-mean-square-deviation (RMSD) of 1.07 Å and an average TM-Score of 0.93 with the modeled designed protein sequences. High sequence recapitulation of 48.7% (59.4%) observed in the design sequences for all (hydrophobic) solvent-inaccessible residues again establish the goodness of the proposed algorithm. A high (93.4%) intra-group recapitulation of hydrophobic residues in the solvent-inaccessible region indicates that the proposed protein design algorithm preserves the core residues in the protein and provides alternative residue combinations in the solvent-accessible regions of the target protein. Furthermore, a COFACTOR-based protein functional analysis shows that the design sequences exhibit altered molecular functionality and introduce new molecular functions compared to the target scaffolds.Communicated by Ramaswamy H. Sarma.
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Proteínas , Motor de Búsqueda , Proteínas/química , Secuencia de Aminoácidos , Simulación por Computador , SolventesRESUMEN
The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics with high neutralization breadth. Here, we characterized a human VH domain, F6, which we generated by sequentially panning large phage-displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that broadly and potently neutralized VOCs including Omicron. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 variants including Omicron and highlight a vulnerable epitope within the spike that may be exploited to achieve broad protection against circulating variants.
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The emergence of SARS-CoV-2 variants necessitates rational assessment of their impact on the recognition and neutralization of the virus by the host cell. We present a comparative analysis of the interactions of Alpha, Beta, Gamma, and Delta variants with cognate molecules (ACE2 and/or furin), neutralizing nanobodies (Nbs), and monoclonal antibodies (mAbs) using in silico methods, in addition to Nb-binding assays. Our study elucidates the molecular origin of the ability of Beta and Delta variants to evade selected antibodies, such as REGN10933, LY-CoV555, B38, C105, or H11-H4, while being insensitive to others including REGN10987. Experiments confirm that nanobody Nb20 retains neutralizing activity against the Delta variant. The substitutions T478K and L452R in the Delta variant enhance associations with ACE2, whereas P681R promotes recognition by proteases, thus facilitating viral entry. The Ab-specific responses of variants highlight how full-atomic structure and dynamics analyses are required for assessing the response to newly emerging variants.
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The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human V H domain, F6, which we generated by sequentially panning large phage displayed V H libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized V H domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC 50 ) of 4.8 nM in vitro . Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.
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Protein design, also known as the inverse protein folding problem, is the identification of a protein sequence that folds into a target protein structure. Protein design is proved as an NP-hard problem. While researchers are working on designing heuristics with an emphasis on new scoring functions, we propose a replica-exchange Monte Carlo (REMC) search algorithm that ensures faster convergence using a greedy strategy. Using biological insights, we construct an evolutionary profile to encode the amino acid variability in different positions of the target protein from its structural homologs. The evolutionary profile guides the REMC search, and the greedy approach confirms appreciable exploration and exploitation of the sequence-structure fitness surface. We allow termination of a simulation trajectory once stagnant situation is detected. A series of sequence and structure level validations establish the goodness of our design. On a benchmark dataset, our algorithm reports an average root-mean-square deviation of 1.21Å between the target and the design proteins when modeled with an existing protein folding software. Besides, our algorithm assures 6.16 times overall speedup. In Molecular Dynamics simulations, we observe that four out of selected five design proteins report better to comparable stability to the corresponding target proteins.
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Algoritmos , Biología Computacional/métodos , Simulación de Dinámica Molecular , Pliegue de Proteína , Proteínas , Método de Montecarlo , Conformación Proteica , Proteínas/química , Proteínas/genética , Proteínas/metabolismoRESUMEN
Insertions/deletions of amino acids in the protein backbone potentially result in altered structural/functional specifications. They can either contribute positively to the evolutionary process or can result in disease conditions. Despite being the second most prevalent form of protein modification, there are no databases or computational frameworks that delineate harmful multipoint deletions (MPD) from beneficial ones. We introduce a positive unlabeled learning-based prediction framework (PROFOUND) that utilizes fold-level attributes, environment-specific properties, and deletion site-specific properties to predict the change in foldability arising from such MPDs, both in the non-loop and loop regions of protein structures. In the absence of any protein structure dataset to study MPDs, we introduce a dataset with 153 MPD instances that lead to native-like folded structures and 7650 unlabeled MPD instances whose effect on the foldability of the corresponding proteins is unknown. PROFOUND on 10-fold cross-validation on our newly introduced dataset reports a recall of 82.2% (86.6%) and a fall out rate (FR) of 14.2% (20.6%), corresponding to MPDs in the protein loop (non-loop) region. The low FR suggests that the foldability in proteins subject to MPDs is not random and necessitates unique specifications of the deleted region. In addition, we find that additional evolutionary attributes contribute to higher recall and lower FR. The first of a kind foldability prediction system owing to MPD instances and the newly introduced dataset will potentially aid in novel protein engineering endeavors.
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Aminoácidos , Proteínas , Ingeniería de Proteínas , Pliegue de Proteína , Proteínas/genéticaRESUMEN
The Viral Protein 35 (VP35), a crucial protein of the Zaire Ebolavirus (EBOV), interacts with a plethora of human proteins to cripple the human immune system. Despite its importance, the entire structure of the tetrameric assembly of EBOV VP35 and the means by which it antagonizes the autophosphorylation of the kinase domain of human protein kinase R (PKRK) is still elusive. We consult existing structural information to model a tetrameric assembly of the VP35 protein where 93% of the protein is modeled using crystal structure templates. We analyze our modeled tetrameric structure to identify interchain bonding networks and use molecular dynamics simulations and normal-mode analysis to unravel the flexibility and deformability of the different regions of the VP35 protein. We establish that the C-terminal of VP35 (VP35C) directly interacts with PKRK to prevent it from autophosphorylation. Further, we identify three plausible VP35C-PKRK complexes with better affinity than the PKRK dimer formed during autophosphorylation and use protein design to establish a new stretch in VP35C that interacts with PKRK. The proposed tetrameric assembly will aid in better understanding of the VP35 protein, and the reported VP35C-PKRK complexes along with their interacting sites will help in the shortlisting of small molecule inhibitors.
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Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Proteínas de la Nucleocápside , Proteínas ViralesRESUMEN
Nonsynonymous single-nucleotide polymorphisms often result in altered protein stability while playing crucial roles both in the evolution process and in the development of human diseases. Prediction of change in the thermodynamic stability due to such missense mutations will help in protein engineering endeavors and will contribute to a better understanding of different disease conditions. Here, we develop a machine-learning-based framework, viz., ProTSPoM, to estimate the change in protein thermodynamic stability arising out of single-point mutations (SPMs). ProTSPoM outperforms existing methods on the S2648 and S1925 databases and reports a Pearson correlation coefficient of 0.82 (0.88) and a root-mean-squared-error of 0.92 (1.06) kcal/mol between the predicted and experimental ΔΔG values on the long-established S350 (tumor suppressor p53 protein) data set. Further, we estimate the change in thermodynamic stability for all possible SPMs in the DNA binding domain of the p53 protein. We identify single-nucleotide polymorphisms in p53 which are plausibly detrimental to its structural integrity and interaction affinity with the DNA molecule. ProTSPoM with its reliable estimates and time-efficient prediction is well suited to be integrated with existing protein engineering techniques. The ProTSPoM web server is accessible at http://cosmos.iitkgp.ac.in/ProTSPoM/.
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Mutación Puntual , Proteína p53 Supresora de Tumor , Humanos , Mutación , Estabilidad Proteica , Termodinámica , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Epithelial to mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling, are the two elemental processes promoting glioblastoma (GBM). In the present work we propose a mechanistic modelling of GBM and in process establish a hypothesis elucidating critical crosstalk between heat shock proteins (HSPs) and matrix metalloproteinases (MMPs) with synergistic upregulation of EMT-like process and ECM remodeling. METHODS: The interaction and the precise binding site between the HSP and MMP proteins was assayed computationally, in-vitro and in GBM clinical samples. RESULTS: A positive crosstalk of HSP27 with MMP-2 and MMP-9 was established in both GBM patient tissues and cell-lines. This association was found to be of prime significance for ECM remodeling and promotion of EMT-like characteristics. In-silico predictions revealed 3 plausible interaction sites of HSP27 interacting with MMP-2 and MMP-9. Site-directed mutagenesis followed by in-vitro immunoprecipitation assay (IP) with 3 mutated recombinant HSP27, confirmed an interface stretch containing residues 29-40 of HSP27 to be a common interaction site for both MMP-2 and MMP-9. This was further validated with in-vitro IP of truncated (sans AA 29-40) recombinant HSP27 with MMP-2 and MMP-9. CONCLUSION: The association of HSP27 with MMP-2 and MMP-9 proteins along with the identified interacting stretch has the potential to contribute towards drug development to inhibit GBM infiltration and migration. GENERAL SIGNIFICANCE: Current findings provide a novel therapeutic target for GBM opening a new horizon in the field of GBM management.