Allosteric and Energetic Remodeling by Protein Domain Extensions
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PROVIDER: PRJNA1276451 | ENA |
REPOSITORIES: ENA
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Project description:Allosteric and Energetic Remodeling by Protein Domain Extensions
| PRJEB90076 | ENA
Project description:Many functions of proteins are performed by independently folding structural units called domains. The structures of domains are conserved during evolution but they are not identical. For example, the >270 human PDZ domains vary in the number of secondary structure elements and in the length of loops. An important but largely unexplored question is the impact of these extensions on protein energy landscapes: beyond any immediate functional effects, do extensions also alter the consequences of perturbations elsewhere in the domain, altering the potential for regulation and evolvability? Here we perform massively parallel energetic measurements on a model human PDZ domain to directly and comprehensively answer this question. In total we quantify the binding to a ligand and abundance of ~190,000 protein variants to quantify free energy changes for mutations throughout the canonical domain fold and ~7,000 energetic couplings between these mutations and the two domain extensions, both alone and in combination. We find that both extensions—one structured and one more dynamic—substantially and specifically re-shape the energy landscape of the domain, with the removal of an ɑ-helix altering the energetic consequences of 424 mutations in 54 sites on fold stability and 420 mutations in 56 sites on binding to a ligand. These changes to the energy landscape alter the effects of 330 allosteric mutations, including at solvent-accessible surface sites. Extending or pruning the domain therefore reshapes its energetic and allosteric landscape, adding and removing opportunities for the allosteric control of protein function.
2025-07-25 | GSE299757 | GEO
Project description:Allosteric communication between distant sites in proteins is central to nearly all biological regulation but still poorly characterised for most proteins, limiting conceptual understanding, biological engineering and allosteric drug development. Typically only a few allosteric sites are known in model proteins, but theoretical, evolutionary and some experimental studies suggest they may be much more widely distributed. An important reason why allostery remains poorly characterised is the lack of methods to systematically quantify long-range communication in diverse proteins. Here we address this shortcoming by developing a method that uses deep mutational scanning to comprehensively map the allosteric landscapes of protein interaction domains. The key concept of the approach is the use of ‘multidimensional mutagenesis’: mutational effects are quantified for multiple molecular phenotypes—here binding and protein abundance—and in multiple genetic backgrounds. This is an efficient experimental design that allows the underlying causal biophysical effects of mutations to be accurately inferred en masse by fitting thermodynamic models using neural networks. We apply the approach to two of the most common human protein interaction domains, an SH3 domain and a PDZ domain, to produce the first global atlases of allosteric mutations for any proteins. Allosteric mutations are widely dispersed with extensive long-range tuning of binding affinity and a large mutational target space of network-altering ‘edgetic’ variants. Mutations are more likely to be allosteric closer to binding interfaces, at Glycines in secondary structure elements and at particular sites including a chain of residues connecting to an opposite surface in the PDZ domain. This general approach of quantifying mutational effects for multiple molecular phenotypes and in multiple genetic backgrounds should allow the energetic and allosteric landscapes of many proteins to be rapidly and comprehensively mapped.
2021-09-13 | GSE184042 | GEO
Project description:Global mapping of the energetic and allosteric landscapes of protein binding domains
| PRJNA762874 | ENA
Project description:The so-called allosteric and torpedo models have been used for the past thirty years to explain how transcription terminates on protein-coding genes. The former invokes conformational changes in the transcription complex and the latter involves degradation of the downstream product of poly(A) signal (PAS) processing. Here, we describe a single mechanism incorporating features of both models. We show that CPSF73 is indispensable for transcriptional termination on protein-coding and its loss causes profound read-through genome-wide. CPSF73 functions upstream of allosteric modifications to the elongation complex that cause Pol II to slow down after the PAS. This state is enriched by rapid depletion of XRN2 and promoted by protein phosphatase 1 (PP1), the inhibition of which confers runaway read-through in the absence of XRN2. These allosteric changes facilitate XRN2-dependent termination, by aiding its capture of Pol II, rather than constituting a termination pathway in themselves. Our experiments unify the long-standing allosteric and torpedo models for transcriptional termination.
2020-01-21 | GSE137727 | GEO
Project description:Enzymes catalyze the reactions of life and are the targets of nearly all small molecule drugs. Most drugs inhibit enzymes by binding to conserved active sites, causing problems of specificity and toxicity. Targeting regulatory allosteric sites can increase specificity, overcome drug resistance and tune or activate activity.However, the vast majority of enzymes have no known allosteric sites and methods do not exist to globally identify or predict them.Here we present a general and fast method to globally chart allosteric communication in enzymes and apply it to the Src protein kinase to produce the first comprehensive map of negative and positive allosteric control of enzymatic activity. Allosteric control of Src is pervasive, anisotropic and distance dependent, but fairly predictable from simple sequence and structural features. The comprehensive allosteric map enables unbiased identification of all the allosterically active surface pockets of the Src kinase for the development of activatory and inhibitory drugs.This general approach can be used to chart global allosteric maps of many kinases, enzymes, and other biochemical activities important for medicine and biotechnology.
2024-04-27 | GSE247740 | GEO
Project description:Intrauterine growth restriction (IUGR) affects 7-10% of pregnancies and is associated with cardiovascular remodeling and dysfunction which persists into adulthood. The underlying subcellular remodeling and cardiovascular programming events are still poorly documented. Cardiac muscle is central in the fetal adaptive mechanism to IUGR given its high energetic demands. The energetic homeostasis depends on the correct interaction of several molecular pathways and the adequate arrangement of intracellular energetic units (ICEUs), where mitochondria interact with the contractile machinery and the main cardiac ATPases to enable a quick and efficient energy transfer. We studied subcellular cardiac adaptations to IUGR in an experimental rabbit model. We evaluated the ultrastructure of ICEUs with transmission electron microscopy and observed an altered spatial arrangement in IUGR, with significant increases in cytosolic space between mitochondria and myofilaments. A global decrease of mitochondrial density was also observed. In addition, we conducted a global gene expression profile by advanced bioinformatics tools to assess the expression of genes involved in the cardiomyocyte energetic metabolism, and identified four gene modules with a coordinated over-representation in IUGR: oxygen homeostasis (GO: 0032364), mitochondrial respiratory chain complex I (GO:0005747), oxidative phosphorylation (GO: 0006119) and NADH dehydrogenase activity (GO:0003954). These findings might contribute to changes in energetic homeostasis in IUGR. The potential persistence and role of these changes in long term cardiovascular programming deserves further investigation. In this study, we used gene expression arrays to investigate differences between two experimental groups: IUGR and control. The design includes paired samples.
2013-10-30 | E-GEOD-37860 | biostudies-arrayexpress
Project description:Numerous missense mutations cause misfolding and premature degradation of ATP-binding cassette (ABC)-transporters-transporters, accounting for several human conformational diseases with poorly under-stood molecular mechanisms. Recent breakthroughs in small molecule combination therapy led transformative improvement of patients’ outlook in cystic fibrosis (CF), caused by several mutations, including the most prevalent deletion of the F508 (delF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, a member of the large ABC-transporter superfamily. By relying on hydrogen deuterium exchange mass spectrometry (HDX-MS), molecular dynamic simulations and biochemical techniques, we demonstrate that the recently approved pharmacophores (VX-445 [elexacaftor] and/or VX-809 [lumacaftor]) mechanism of action relies on a complex network of dynamic inter-domain allosteric interactions to restore the post-translational coupled domain folding and the final fold stability of mutant CFTRs.
2024-01-26 | PXD042481 | Pride
Project description:Intrauterine growth restriction (IUGR) affects 7-10% of pregnancies and is associated with cardiovascular remodeling and dysfunction which persists into adulthood. The underlying subcellular remodeling and cardiovascular programming events are still poorly documented. Cardiac muscle is central in the fetal adaptive mechanism to IUGR given its high energetic demands. The energetic homeostasis depends on the correct interaction of several molecular pathways and the adequate arrangement of intracellular energetic units (ICEUs), where mitochondria interact with the contractile machinery and the main cardiac ATPases to enable a quick and efficient energy transfer. We studied subcellular cardiac adaptations to IUGR in an experimental rabbit model. We evaluated the ultrastructure of ICEUs with transmission electron microscopy and observed an altered spatial arrangement in IUGR, with significant increases in cytosolic space between mitochondria and myofilaments. A global decrease of mitochondrial density was also observed. In addition, we conducted a global gene expression profile by advanced bioinformatics tools to assess the expression of genes involved in the cardiomyocyte energetic metabolism, and identified four gene modules with a coordinated over-representation in IUGR: oxygen homeostasis (GO: 0032364), mitochondrial respiratory chain complex I (GO:0005747), oxidative phosphorylation (GO: 0006119) and NADH dehydrogenase activity (GO:0003954). These findings might contribute to changes in energetic homeostasis in IUGR. The potential persistence and role of these changes in long term cardiovascular programming deserves further investigation.
2013-10-30 | GSE37860 | GEO
Project description:The NAD hydrolase (NADase) SARM1 acts as a central executioner of programmed axon death and is a possible therapeutic target for neurodegenerative disorders. While orthosteric inhibitors of SARM1 have been described, this multi-domain enzyme is also subject to intricate forms of autoregulation, suggesting the potential for allosteric modes of inhibition. Previous studies have identified multiple cysteine residues that support SARM1 activation and catalysis, but which of these cysteines, if any, might be selectively targetable by electrophilic small molecules remains unknown. Here we describe the chemical proteomic discovery of a series of tryptoline acrylamides that site-specifically and stereoselectively modify cysteine-311 (C311) in the non-catalytic, autoregulatory armadillo repeat (ARM) domain of SARM1. These covalent compounds inhibit the NADase activity of WT-SARM1, but not C311A or C311S SARM1 mutants, show a high degree of proteome-wide selectivity for SARM1_C311, and stereoselectively block vincristine- and vacor-induced neurite degeneration in primary rodent dorsal root ganglion neurons. Our findings describe selective, covalent inhibitors of SARM1 targeting an allosteric cysteine, pointing to a potentially attractive therapeutic strategy for axon degeneration-dependent forms of neurological disease.
2022-08-22 | PXD035400 | Pride