Allosteric Activation Research Articles

Mechanism of allosteric activation in human mitochondrial ClpP protease

Human ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition leads to the accumulation of damaged respiratory chain subunits and cell death. Conversely, hyperactivating ClpP with small-molecule activators, such as the recently discovered ONC201, disrupts mitochondrial protein degradation and impairs respiration in cancer cells. Despite its critical role in human health, the mechanism underlying the structural and functional properties of human ClpP remains elusive. Notably, human ClpP is paradoxically activated by active-site inhibitors. All available structures of human ClpP published to date are in the inactive compact or compressed states, surprisingly even when ClpP is bound to an activator molecule such as ONC201. Here, we present structures of human mitochondrial ClpP in the active extended state, including a pair of structures where ClpP is bound to an active-site inhibitor. We demonstrate that amino acid substitutions in the handle region (A192E and E196R) recreate a conserved salt bridge found in bacterial ClpP, stabilizing the extended active state and significantly enhancing ClpP activity. We elucidate the ClpP activation mechanism, highlighting a hormetic effect where substoichiometric inhibitor binding triggers an allosteric transition that drives ClpP into its active extended state. Our findings link the conformational dynamics of ClpP to its catalytic function and provide high-resolution structures for the rational design of potent and specific ClpP inhibitors, with implications for targeting AML and other disorders with ClpP involvement.

Thiamine, gastrointestinal beriberi and acetylcholine signaling

Research has highlighted numerous detrimental consequences of thiamine deficiency on digestive function. These range from impaired gastric and intestinal motility to aberrant changes in pancreatic exocrine function, gastric acidity and disturbances in gut barrier integrity and inflammation. Thiamine and its pharmacological forms, as a primary or adjunctive therapy, have been shown to improve symptoms such as nausea, constipation, dysphagia and intestinal dysmotility, in both humans and animals. This review aims to explore molecular mechanisms underlying the therapeutic action of thiamine in gastrointestinal dysfunction. Our analysis demonstrates that thiamine insufficiency restricted to the gastrointestinal system, i.e., lacking well-known symptoms of dry and wet beriberi, may arise through (i) a disbalance between the nutrient influx and efflux in the gastrointestinal system due to increased demands of thiamine by the organism; (ii) direct exposure of the gastrointestinal system to oral drugs and gut microbiome, targeting thiamine-dependent metabolism in the gastrointestinal system in the first line; (iii) the involvement of thiamine in acetylcholine (ACh) signaling and cholinergic activity in the enteric nervous system and non-neuronal cells of the gut and pancreas, employing both the coenzyme and non-coenzyme actions of thiamine. The coenzyme action relies on the requirement of the thiamine coenzyme form – thiamine diphosphate – for the production of energy and acetylcholine (ACh). The non-coenzyme action involves participation of thiamine and/or derivatives, including thiamine triphosphate, in the regulation of ACh synaptic function, consistent with the early data on thiamine as a co-mediator of ACh in neuromuscular synapses, and in allosteric action on metabolic enzymes. By examining the available evidence with a focus on the gastrointestinal system, we deepen the understanding of thiamine’s contribution to overall gastrointestinal health, highlighting important implications of thiamine-dependent mechanisms in functional gastrointestinal disorders.

Novel Spiro-Barbiturates Can Reverse the Action of General Anesthetics on the GABAAR.

GABAARs are pentameric ligand-gated ion channels that play a major role in mediating inhibition in the CNS. They are the target of many widely used positive allosteric modulators (PAMs) of GABAARs such as general anesthetics, sedatives, antiepileptics, and anxiolytics. However, close structural analogs of these PAMs are negative allosteric modulators that cause excitation. Comparison of the SAR of inhibitory and excitatory barbiturates suggested that conformationally constrained spiro-analogs of phenobarbital might have intermediate allosteric activity. More than 50 spiro-analogs were synthesized and characterized for their ability to enhance desensitization and reverse the action of anesthetics. A number of these reversed the action of anesthetics without having any action on GABA-induced desensitization. These constitute a new class of GABA-ergic drugs that are null allosteric ligands. They offer the potential to reverse the sedative action of current PAMs and modulate the behavior of diseases resulting from mutations in GABAAR subunits.

Redox regulation and dynamic control of brain-selective kinases BRSK1/2 in the AMPK family through cysteine-based mechanisms.

In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications, including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intramolecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide-mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.

New homocysteine consumption assay for high-throughput screening of human cystathionine-β-synthase.

New homocysteine consumption assay for high-throughput screening of human cystathionine-β-synthase.

The BK channel-NS1619 agonist complex reveals molecular insights on allosteric activation gating.

BK channel activation by NS1619 reveals key binding interactions, offering insights for designing targeted therapeutic agents.

ERK Allosteric Activation: The Importance of Two Ordered Phosphorylation Events.

ERK Allosteric Activation: The Importance of Two Ordered Phosphorylation Events.

Conformational dynamics and activation of membrane-associated human Group IVA cytosolic phospholipase A 2 (cPLA 2 ).

Cytosolic phospholipase A 2 (cPLA 2 ) associates with membranes where it hydrolyzes phospholipids containing arachidonic acid to initiate an inflammatory cascade. All-atom molecular dynamics simulations were employed to understand the activation process when cPLA 2 associates with the endoplasmic reticulum (ER) membrane of macrophages where it acts. We found that membrane association causes the lid region of cPLA 2 to undergo a closed-to-open state transition that is accompanied by the sideways movement of loop 495-540, allowing the exposure of a cluster of lysine residues (K488, K541, K543, and K544), which binds the allosteric activator PIP 2 in the membrane. The active site of the open form of cPLA 2 , containing the catalytic dyad residues S228 and D549, exhibited a three-fold larger cavity than the closed form of cPLA 2 in aqueous solution. These findings provide mechanistic insight as to how cPLA 2 ER membrane association promotes major transitions between conformational states critical to allosteric activation and enzymatic phospholipid hydrolysis.

Cytoplasmic Mg2+ supersedes carbon source preference to dictate Salmonella metabolism

Glucose is the preferred carbon source of most studied microorganisms. However, we now report that glucose loses preferred status when the intracellular pathogen Salmonella enterica serovar Typhimurium experiences cytoplasmic magnesium (Mg2+) starvation. We establish that this infection-relevant stress drastically reduces synthesis of cyclic adenosine monophosphate (cAMP), the allosteric activator of the cAMP receptor protein (CRP), master regulator of carbon utilization. The resulting reduction in cAMP concentration, which is independent of carbon source, decreases transcription of CRP-cAMP-activated carbon utilization genes, hinders carbon source uptake, and restricts metabolism, rendering wild-type bacteria phenotypically CRP-. A cAMP-independent allele of CRP overcame the transcriptional, uptake, and metabolic restrictions caused by cytoplasmic Mg2+ starvation and significantly increased transcription of the glucose uptake gene when S. Typhimurium was inside murine macrophages. The reduced preference for glucose exhibited by S. Typhimurium inside macrophages reflects that transcription of the glucose uptake gene requires higher amounts of active CRP-cAMP than transcription of uptake genes for preferred carbon sources, such as gluconate and glycerol. By reducing CRP-cAMP activity, low cytoplasmic Mg2+ alters carbon source preference, adjusting metabolism and growth.

Uncovering the Role of Distal Regions in PDK1 Allosteric Activation

Uncovering the Role of Distal Regions in PDK1 Allosteric Activation

Functional Spectrum of USP7 Pathogenic Variants in Hao-Fountain Syndrome: Insights into the Enzyme's Activity, Stability, and Allosteric Modulation.

Hao-Fountain syndrome is a rare neurodevelopmental disorder caused by mutations in the de-ubiquitinating enzyme USP7 (Ubiquitin Specific Protease 7). Due to the novelty of the disease and its poorly understood molecular mechanisms, treatments for the syndrome are currently lacking. This study examines the effects of 11 patient-derived variants located within the catalytic domain of USP7, focusing on their impact on the enzyme's activity, thermodynamic stability, and substrate recognition. Our findings reveal a spectrum of functional consequences, ranging from complete inactivation to hyperactivation of USP7. Notably, we identify a specific subset of pathogenic variants whose catalytic activity can be significantly boosted using a novel allosteric activator. These results provide the first insight into USP7 malfunction in Hao-Fountain syndrome-linked variants and pave the way for improved prognostic approaches and targeted treatments in the future.

Impact of Cancer-Associated PKM2 Mutations on Enzyme Activity and Allosteric Regulation: Structural and Functional Insights into Metabolic Reprogramming.

Mammalian pyruvate kinase M2 (PKM2) is a key regulator of glycolysis and is highly expressed in proliferative tissues including tumors. Mutations in PKM2 have been identified in various cancers, but their effects on enzyme activity and regulation are not fully understood. This study investigates the structural and functional effects of cancer-associated PKM2 mutations on enzyme kinetics, allosteric regulation, and oligomerization. Using computational modeling, X-ray crystallography, and biochemical assays, we demonstrated how these mutations impact PKM2 activity, substrate binding, and allosteric activation via fructose-1,6-bisphosphate (FBP), contributing to altered enzyme function. In this study, we characterized four cancer-associated PKM2 mutations (P403A, C474S, R516C, and L144P) using computational, structural, and biochemical approaches. Computational modeling revealed disruptions in allosteric signaling pathways, particularly affecting the communication between regulatory sites and the active site. X-ray crystallography demonstrated local conformational changes in the hinge and FBP-binding regions, leading to a shift from the active tetrameric state to a less active dimeric state, particularly in the C474S and R516C mutants. The mutants exhibited reduced maximal velocity, reduced substrate affinity, and altered activation by the allosteric activator fructose-1,6-bisphosphate (FBP). Under alkaline pH conditions, mimicking the tumor microenvironment, these mutations further destabilized the PKM2 oligomeric state, favoring the formation of lower-order species. Our findings suggest that PKM2 is highly sensitive to mutations, and these alterations may contribute to metabolic reprogramming in cancer cells by impairing its enzymatic regulation.

The EC2 domains of tetraspanins CD9, CD81, and CD151 bind to the allosteric site of integrins (site 2) and activate integrins αvβ3, α5β1 and α4β1 in a biphasic manner.

Previous studies showed that tetraspanins activate integrins, but the mechanism of this action is unclear. We previously showed that the extracellular-2 (EC2) domains of CD9, CD81, and CD151 bind to the classical RGD-binding site (site 1) of integrin αvβ3, suggesting that they are integrin ligands. We showed that several inflammatory cytokines (e.g., CX3CL1, CXCL12, CCL5, and CD40L) bind to the allosteric site (site 2) of integrins, which is distinct from site 1, and activate integrins (allosteric activation). 25-hydroxycholesterol, a major inflammatory lipid mediator, is known to bind to site 2 and induce inflammatory signals, suggesting that site 2 plays a role in inflammatory signaling. We hypothesized that the EC2 domains activate integrins by binding to site 2. Here we describe that docking simulation predicted that CD81 EC2 binds to site 2 of αvβ3 and more strongly to site 2 of α5β1. Peptide from site 2 bound to isolated EC2 domains, suggesting that the EC2 domains bind to site 2. The EC2 domains only weakly activated αvβ3 but more efficiently activated cell surface integrins α5β1 and α4β1 on the cell surface. These results are consistent with the previous findings that these tetraspanins preferentially interact with β1 integrins. The integrin activation by the EC2 domains was increased at low EC2 concentrations and reduced as EC2 concentrations increased (biphasic), which is consistent with the findings that the EC2 domains bind to two sites (site 1 and 2). We propose that the EC2 binding to site 2 is a novel target for drug discovery.

Mitapivat: A Novel Drug Discovery for the Treatment of Hereditary Hemolytic Anaemias

: Mitapivat (AG-348) is a novel, first-in-class oral small-molecule allosteric activator of the pyruvate kinase enzyme. Mitapivat has been shown to significantly upregulate both wild-type and numerous mutant forms of erythrocyte pyruvate kinase (PKR), increasing adenosine triphosphate (ATP) production and reducing levels of 2,3-diphosphoglycerate. Given this mechanism, mitapivat has been evaluated in clinical trials in a wide range of hereditary hemolytic anaemias, including pyruvate kinase deficiency (PKD), sickle cell disease, and thalassemia. : The technique entails searching numerous search engines, such as PubMed, Science Direct, and Sci Finder, for relevant citations to the current subject matter. This is done in order to obtain the data that is required. In relation to medicine, mitapivat has been examined for its ability to cure a wide variety of inherited haemolytic anaemias in clinical investigations. Some examples of these conditions include pyruvate kinase deficiency (PKD), sickle cell disease, and thalassemias. : It has been demonstrated that mitapivat is both safe and effective in treating adults with PKD in two phases III clinical trials, and the development of the medicine is very close to being finished. Based on these findings, mitapivat may end up becoming the very first medication in the history of the world to receive regulatory approval. : Allosteric activator of pyruvate kinase mitapivat has shown promise in treating various hereditary hemolytic anemias, including sickle cell disease, PKD, and alpha- and beta-thalassemia.

Structural basis for the allosteric activation of Lon by the heat shock protein LarA

Lon is a conserved AAA+ (ATPases associated with diverse cellular activities) proteolytic machine that plays a key regulatory role in cells under proteotoxic stress. Lon-mediated proteolysis can be stimulated by either the unfolded or specific protein substrates accumulated under stress conditions. However, the molecular basis for this substrate-controlled proteolysis remains unclear. Here, we have found that the heat shock protein LarA, a recently discovered Lon substrate and allosteric activator, binds to the N-terminal domain (NTD) of Lon. The crystal structure of the LarA-NTD complex shows that LarA binds to a highly conserved groove in the NTD through the terminal aromatic residue of its C-terminal degron. Crystallographic and biochemical evidence further reveals that this binding exposes the hydrophobic core of LarA, which can bind a leucine residue and promote local protein unfolding. These results define the mechanistic role of the NTD in regulating Lon-mediated proteolysis in response to varying cellular conditions.

Programmable Split DNAzyme Modulators via Allosteric Cooperative Activation for mRNA Electrochemiluminescence Biosensing.

DNAzymes, known for their programmability, stability, and cost-effectiveness, are powerful tools for signal transduction in complex biological systems. However, their application in responding to target effectors is often hindered by limited catalytic efficiency and susceptibility to unintended activation. Here we propose an allosteric cooperative activation strategy to program a split DNAzyme modulator (STATER) that enables sensitive and accurate electrochemiluminescence (ECL) biosensing of interleukin-6 (IL-6) mRNA. Our design features a STATER that leverages a DNA tetrahedron as a central scaffold, equipped with two pairs of T-shaped hairpin probes (TP) and helper hairpin probes (HP). Specifically, the TP contains two apurinic/apyrimidinic endonuclease 1 (APE1) recognition sites, an IL-6 mRNA recognition region, and a partzyme fragment, while the HP contains a corresponding paired partzyme fragment. Unlike conventional DNAzyme modulators that rely on single effector activation, the STATER integrates an allosteric cooperative activation mechanism, which ensures that all preblocked components are synergistically activated and assembled within a confined space, facilitating rapid and specific reconstruction of the DNAzyme's catalytic active domain. Furthermore, upon cooperative recognition by APE1 and IL-6 mRNA, two inactive partzymes undergo an allosteric assembly via a toehold exchange displacement reaction, switching on the cleavage reactivity of STATER. This mechanism enables the establishment of an activation threshold for IL-6 mRNA, thereby minimizing nonspecific activation in complex scenarios. Our studies demonstrate that the STATER exhibits outstanding sensitivity and selectivity for IL-6 mRNA detection using the supramolecular gold nanoclusters network-based ECL platform. The biosensor provides a linear detection span from 1 × 10-13 to 1 × 10-7 M, with a limit of detection as low as 3.26 × 10-14 M, highlighting STATER's potential for detecting various analytes in complex biological systems.

Conversion of glutamate into proline by the leucine analog BCH enhances biphasic insulin secretion in pancreatic β-cells.

Biphasic insulin secretion, which fails in Type 2 Diabetes, can be provoked by various nutrient stimuli, glucose being the superior physiological one. To identify pathways that may play a role in β-cell stimulus-secretion coupling, we compared β-cell and islet functional, secretory, and metabolic responses to glucose and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), a leucine analog, that acts as an allosteric activator of glutamate dehydrogenase (GDH). We employed a range of techniques, including insulin secretion assays, mitochondrial activity measurements, ATP/ADP ratio assessments, and cytosolic Ca2+ level quantifications. Metabolomics was used to analyze cellular metabolite profiles in response to glucose and BCH. Additionally, we investigated the role of proline synthesis by silencing ALDH18A1, encoding proline 5-carboxylate (P5C) synthase, in both clonal β-cells and human islets. BCH and glucose similarly induced a biphasic insulin response in INS-1 832/13 cells, parallelled by increased mitochondrial activity and raised ATP/ADP ratios, plasma membrane depolarization, and elevated cytosolic Ca2+ levels. Metabolomics revealed that proline levels increased significantly only in BCH-stimulated β-cells. Silencing ALDH18A1 disrupted insulin secretion in response to both glucose and BCH, accompanied by reduced cytosolic Ca2+ levels, ATP/ADP ratios, and mitochondrial activity. Our findings demonstrated that BCH-induced activation of GDH leads to the conversion of glutamate into proline, which apparently enhances β-cell stimulus-secretion coupling. This work identifies a previously unrecognized role of proline metabolism in β-cell function and provides novel insights into the complex regulation of insulin secretion.

Balanced plant helper NLR activation by a modified host protein complex.

Nucleotide-binding leucine-rich repeat (NLR) receptors play crucial roles in plant immunity by sensing pathogen effectors1. In Arabidopsis, certain sensor NLRs function as NADases to catalyse the production of second messengers2,3, which can be recognized by enhanced disease susceptibility 1 (EDS1) with its partner senescence-associated gene 101 (SAG101), to activate helper NLR N requirement gene 1 (NRG1)4. A cryoelectron microscopy structure shows that second-messenger-activated EDS1-SAG101 mainly contacts the leucine-rich repeat domain of NRG1A to mediate the formation of an induced EDS1-SAG101-NRG1A complex. Structural comparisons show that binding of a second messenger induces conformational changes in EDS1-SAG101, which are recognized by NRG1A, leading to its allosteric activation. We further show that an inhibitory NRG1 family member, NRG1C, efficiently outcompetes NRG1A for binding to second-messenger-activated EDS1-SAG101. These findings uncover mechanisms for NRG1A activation through itsrecognition of a modified host EDS1-SAG101 complex, and NRG1A inhibition by NRG1C through sequestration of the activated EDS1-SAG101, thus shedding light on the activation and constraint of a central plant immune response system.

Integrated Computational Analysis of C-2 Substituted Pyrazolopyrimidine and Amide Isosteres ALLINI: 3D-QSAR, Molecular Docking, and ADMET Studies.

The rapid increase in incidences of drug resistance and off-target toxicity in the case of Human Immunodeficiency Virus (HIV) has increased the demand for drugs with fewer side effects. HIV-1 Integrase (IN) is a promising target that helps integrate viral DNA with human DNA. It acts as a target for strand transfer inhibitors. However, the emergence of resistant mutations in the proteins necessitates the exploration of potent allosteric drugs. The allosteric integrase inhibitors (ALLINI) that interrupt the association of the integrase binding domain of the lens epithelium growth factor (LEDGF/p75) and LEDGF/p75 binding site of the IN are more promising as they hinder site specificity and viral replication. In this study, a 3D-QSAR, molecular docking, and ADMET were carried out to investigate the binding of the C2-pyrazolopyrimidine amides and amide isosteres. The 3D-QSAR model was developed using a series of 24 C-2 substituted pyrazolopyrimidine and amide isosteres. A statistically significant model was constructed, showing the determination coefficient (r2) and five-fold cross-validation (q2) at 0.946 and 0.506, respectively. Furthermore, the contour maps of the electrostatic potential and van der Waals coefficient provided structural modifications in the features to improve the inhibitory activity. A molecular docking study was also performed to check the binding of the compounds to the LEDGF/p75 binding site of the IN, along with ADMET evaluation. The outcome of the study will help to prepare the potent molecules with enhanced allosteric inhibitory activity.

Structures of the cyanobacterial nitrogen regulators NtcA and PipX complexed to DNA shed light on DNA binding by NtcA and implicate PipX in the recruitment of RNA polymerase.

The CRP-FNR (cAMP receptor protein-fumarate/nitrate reductase regulator) superfamily of transcriptional regulators includes the cyanobacterial master regulator NtcA, which orchestrates large responses of cyanobacteria to nitrogen scarcity. NtcA uses as allosteric activator 2-oxoglutarate (2OG), a signal of nitrogen poorness and carbon richness, and binds a co-activating protein (PipX) that shuttles between the signaling protein PII and NtcA depending on nitrogen richness, thus connecting PII signaling and gene expression regulation. Here, combining structural (X-ray crystallography of six types of crystals including NtcA complexes with DNA, 2OG, and PipX), modeling, and functional [electrophoretic mobility shift assays and bacterial two-hybrid (BACTH)] studies, we clarify the reasons for the exquisite specificity for the binding of NtcA to its target DNA, its mechanisms of activation by 2OG, and its co-activation by PipX. Our crystal structures of PipX-NtcA-DNA complexes prove that PipX does not interact with DNA, although it increases NtcA-DNA contacts, and that it stabilizes the active, DNA-binding-competent conformation of NtcA. Superimposition of this complex on a very recently reported cryo-electron microscopy structure of NtcA in a transcription activity complex with RNA polymerase (RNAP), shows that PipX binding helps recruit RNAP by PipX interaction with RNAP, particularly with its gamma and sigma (region 4) subunits, a structural prediction supported here by BACTH experiments.