Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A common allosteric site and mechanism in caspases.

ABSTRACT: We present a common allosteric mechanism for control of inflammatory and apoptotic caspases. Highly specific thiol-containing inhibitors of the human inflammatory caspase-1 were identified by using disulfide trapping, a method for site-directed small-molecule discovery. These compounds became trapped by forming a disulfide bond with a cysteine residue in the cavity at the dimer interface approximately 15 A away from the active site. Mutational and structural analysis uncovered a linear circuit of functional residues that runs from one active site through the allosteric cavity and into the second active site. Kinetic analysis revealed robust positive cooperativity not seen in other endopeptidases. Recently, disulfide trapping identified a similar small-molecule site and allosteric transition in the apoptotic caspase-7 that shares only a 23% sequence identity with caspase-1. Together, these studies show a general small-molecule-binding site for functionally reversing the zymogen activation of caspases and suggest a common regulatory site for the allosteric control of inflammation and apoptosis.

SUBMITTER: Scheer JM

PROVIDER: S-EPMC1458511 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Discovery of an allosteric site in the caspases.

Project description:Allosteric regulation of proteins by conformational change is a primary means of biological control. Traditionally it has been difficult to identify and characterize novel allosteric sites and ligands that freeze these conformational states. We present a site-directed approach using Tethering for trapping inhibitory small molecules at sites away from the active site by reversible disulfide bond formation. We screened a library of 10,000 thiol-containing compounds against accessible cysteines of two members of the caspase family of proteases, caspase-3 and -7. We discovered a previously unreported and conserved allosteric site in a deep cavity at the dimer interface 14 A from the active site. This site contains a natural cysteine that, when disulfide-bonded with either of two specific compounds, inactivates these proteases. The allosteric site is functionally coupled to the active site, such that binding of the compounds at the allosteric site prevents peptide binding at the active site. The x-ray crystal structures of caspase-7 bound by either compound demonstrates that they inhibit caspase-7 by trapping a zymogen-like conformation. This approach may be useful to identify new allosteric sites from natural or engineered cysteines, to study allosteric transitions in proteins, and to nucleate drug discovery efforts.

| S-EPMC514654 | biostudies-literature

Allosteric mechanism for site-specific ubiquitination of FANCD2.

Project description:DNA-damage repair is implemented by proteins that are coordinated by specialized molecular signals. One such signal in the Fanconi anemia (FA) pathway for the repair of DNA interstrand crosslinks is the site-specific monoubiquitination of FANCD2 and FANCI. The signal is mediated by a multiprotein FA core complex (FA-CC) however, the mechanics for precise ubiquitination remain elusive. We show that FANCL, the RING-bearing module in FA-CC, allosterically activates its cognate ubiqutin-conjugating enzyme E2 UBE2T to drive site-specific FANCD2 ubiquitination. Unlike typical RING E3 ligases, FANCL catalyzes ubiquitination by rewiring the intraresidue network of UBE2T to influence the active site. Consequently, a basic triad unique to UBE2T engages a structured acidic patch near the target lysine on FANCD2. This three-dimensional complementarity, between the E2 active site and substrate surface, induced by FANCL is central to site-specific monoubiquitination in the FA pathway. Furthermore, the allosteric network of UBE2T can be engineered to enhance FANCL-catalyzed FANCD2-FANCI di-monoubiquitination without compromising site specificity.

| S-EPMC7035956 | biostudies-literature

Evolution of an allosteric "off switch" in apoptotic caspases.

Project description:Caspase-3 is well known as the "executioner" whose activation commits the cell to an apoptotic fate, but low levels of caspase-3 activity also play key roles in development. A new study explains how cells can balance these functions, using biophysical, structural, and computational approaches to demonstrate the mechanism by which phosphorylation of conserved sites on a distal surface loop reduces or abolishes catalytic activity. These results provide new insights into allosteric regulation mechanisms and offer new opportunities for development of caspase-3 modulators.

| S-EPMC5900751 | biostudies-literature

Evidence for a common mechanism of SIRT1 regulation by allosteric activators.

Project description:A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1? and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.

| S-EPMC3799917 | biostudies-literature

A common allosteric mechanism regulates homeostatic inactivation of auxin and gibberellin.

Project description:Allosteric regulation is protein activation by effector binding at a site other than the active site. Here, we show via X-ray structural analysis of gibberellin 2-oxidase 3 (GA2ox3), and auxin dioxygenase (DAO), that such a mechanism maintains hormonal homeostasis in plants. Both enzymes form multimers by interacting via GA4 and indole-3-acetic acid (IAA) at their binding interface. Via further functional analyses we reveal that multimerization of these enzymes gradually proceeds with increasing GA4 and IAA concentrations; multimerized enzymes have higher specific activities than monomer forms, a system that should favour the maintenance of homeostasis for these phytohormones. Molecular dynamic analysis suggests a possible mechanism underlying increased GA2ox3 activity by multimerization-GA4 in the interface of oligomerized GA2ox3s may be able to enter the active site with a low energy barrier. In summary, homeostatic systems for maintaining GA and IAA levels, based on a common allosteric mechanism, appear to have developed independently.

| S-EPMC7195466 | biostudies-literature

A common intracellular allosteric binding site for antagonists of the CXCR2 receptor.

Project description:Background and purposeWe have previously shown that SB265610 (1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea) behaves as an allosteric, inverse agonist at the C-X-C chemokine (CXCR)2 receptor. The aim of this study was to determine whether SB265610, in addition to two other known antagonists, bind to either of the two putative, topographically distinct, allosteric binding sites previously reported in the Literature.Experimental approachTen single point mutations were introduced into the CXCR2 receptor using site-directed mutagenesis. Three CXCR2 antagonists were investigated, SB265610, Pteridone-1 (2-(2,3 difluoro-benzylsulphanyl)-4-((R)-2-hydroxy-1-methyl-ethylamino)-8H-pteridin-7-one) and Sch527123 (2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1enylamino}-benzamide), and the effect of these mutations on their binding affinity and ability to inhibit interleukin-8-stimulated binding of [(35)S]GTPgammaS was examined.Key resultsSeven of the nine mutations introduced into the C-terminal domain and intracellular loops of the receptor produced a significant reduction in affinity at least one of the antagonists tested. Of those seven mutations, three produced a significant reduction in the affinity of all three antagonists, namely K320A, Y314A and D84N. In all but one mutation, the changes observed on antagonist affinity were matched with effects on inhibition of interleukin-8-stimulated [(35)S]GTPgammaS binding.Conclusions and implicationsThese antagonists bind to a common intracellular, allosteric, binding site of the CXCR2 receptor, which has been further delineated. As many of these mutations are close to the site of G protein coupling or to a region of the receptor that is responsible for the transduction of the activation signal, our results suggest a molecular mechanism for the inhibition of receptor activation.

| S-EPMC2850400 | biostudies-literature

Illumination of serotonin transporter mechanism and role of the allosteric site.

Project description:The serotonin transporter (SERT) terminates serotonin signaling by using sodium and chloride gradients to drive reuptake of serotonin into presynaptic neurons and is the target of widely used medications to treat neuropsychiatric disorders. Despite decades of study, the molecular mechanism of serotonin transport, the coupling to ion gradients, and the role of the allosteric site have remained elusive. Here, we present cryo–electron microscopy structures of SERT in serotonin-bound and serotonin-free states, in the presence of sodium or potassium, resolving all fundamental states of the transport cycle. From the SERT-serotonin complex, we localize the substrate-bound allosteric site, formed by an aromatic pocket positioned in the scaffold domain in the extracellular vestibule, connected to the central site via a short tunnel. Together with elucidation of multiple apo state conformations, we provide previously unseen structural understanding of allosteric modulation, demonstrating how SERT binds serotonin from synaptic volumes and promotes unbinding into the presynaptic neurons.

| S-EPMC8635421 | biostudies-literature

Structural insight to mutation effects uncover a common allosteric site in class C GPCRs.

Project description:MotivationClass C G protein-coupled receptors (GPCRs) regulate important physiological functions and allosteric modulators binding to the transmembrane domain constitute an attractive and, due to a lack of structural insight, a virtually unexplored potential for therapeutics and the food industry. Combining pharmacological site-directed mutagenesis data with the recent class C GPCR experimental structures will provide a foundation for rational design of new therapeutics.ResultsWe uncover one common site for both positive and negative modulators with different amino acid layouts that can be utilized to obtain selectivity. Additionally, we show a large potential for structure-based modulator design, especially for four orphan receptors with high similarity to the crystal structures.Availability and implementationAll collated mutagenesis data is available in the GPCRdb mutation browser at http://gpcrdb.org/mutations/ and can be analyzed online or downloaded in excel format.Contactdavid.gloriam@sund.ku.dk.Supplementary informationSupplementary data are available at Bioinformatics online.

| S-EPMC5408886 | biostudies-literature

Small Molecule Active Site Directed Tools for Studying Human Caspases.

Project description:Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes.

| S-EPMC5610424 | biostudies-literature

Modifications to a common phosphorylation network provide individualized control in caspases.

Project description:Caspase-3 activation and function have been well-defined during programmed cell death, but caspase activity, at low levels, is also required for developmental processes such as lymphoid proliferation and erythroid differentiation. Post-translational modification of caspase-3 is one method used by cells to fine-tune activity below the threshold required for apoptosis, but the allosteric mechanism that reduces activity is unknown. Phosphorylation of caspase-3 at a conserved allosteric site by p38-MAPK (mitogen-activated protein kinase) promotes survival in human neutrophils, and the modification of the loop is thought to be a key regulator in many developmental processes. We utilized phylogenetic, structural, and biophysical studies to define the interaction networks that facilitate the allosteric mechanism in caspase-3. We show that, within the modified loop, Ser150 evolved with the apoptotic caspases, whereas Thr152 is a more recent evolutionary event in mammalian caspase-3. Substitutions at Ser150 result in a pH-dependent decrease in dimer stability, and localized changes in the modified loop propagate to the active site of the same protomer through a connecting surface helix. Likewise, a cluster of hydrophobic amino acids connects the conserved loop to the active site of the second protomer. The presence of Thr152 in the conserved loop introduces a "kill switch" in mammalian caspase-3, whereas the more ancient Ser150 reduces without abolishing enzyme activity. These data reveal how evolutionary changes in a conserved allosteric site result in a common pathway for lowering activity during development or a more recent cluster-specific switch to abolish activity.

| S-EPMC5900778 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data