Mechanism Of Allosteric Activation Research Articles

Computational and Functional Analyses of HER2 Mutations Reveal Allosteric Activation Mechanisms and Altered Pharmacologic Effects.

This study identified new oncogenic HER2 allosteric mutations, including ECD mutations that share covalent dimerization as a mechanism of oncogenicity, suggesting the need for novel inhibitors to treat HER2-mutant cancers.

Minimal mechanistic component of proteasome activation and prevention of impairment by pathological oligomers

Impairment of proteasomal function has been implicated in neurodegenerative diseases, justifying the need to understand how the proteasome is activated for protein degradation. Here, using biochemical and structural (cryo‐EM) strategies in both archaeal and mammalian proteasomes, we further determine the HbYX(‐motif)‐dependent mechanism of proteasomal activation used by multiple proteasome‐activating complexes including the 19S Particle. We identify multiple proteasome α subunit residues involved in HbYX‐dependent activation, a point mutation that activates the proteasome by partially mimicking a HbYX‐bound state, and conformational changes involved in gate‐opening with a 2.1A structure. Through an iterative process of peptide synthesis, we successfully design a HbYX‐like dipeptide mimetic as a robust tool to elucidate how the motif autonomously activates the proteasome. The mimetic induces near complete gate‐opening at saturating concentration, activating mammalian proteasomal degradation of peptides and proteins. Findings using our peptide mimetic suggest the HbYX‐dependent mechanism requires cooperative binding in at least two intersubunit pockets of the α ring. Collectively, the results presented here unambiguously demonstrate the lone role of the HbYX tyrosine in the allosteric mechanism of proteasome activation and offer proof of concept for the robust potential of HbYX‐like small molecules to activate the proteasome.

Biochemical Characterization of Starch Excess4 from Storage Crops

Starch is a water‐insoluble branched polymer of glucose. Its degradation requires the concerted action of glucan phosphatases and glucan dikinases. Reversible starch phosphorylation allows for the solubilization of the starch surface, allowing for amylase to degrade starch into maltose units. However, our understanding is limited on how these enzymes contribute to starch degradation in storage crops such as potato and cassava. In this study, we biochemically characterized cassava and potato SEX4 enzymes using enzymatic assays and differential scanning fluorometry. We determined the kinetics of SEX4 using both generic and physiologically relevant substrates. We also explored the utilization of these glucan phosphatase orthologs to enhance starch degradation in‐vitro. Our findings reveal that cassava and potato SEX4 show similar allosteric activation mechanisms but varying abilities to degrade starch under in‐vitro conditions.

Structure of a Janus kinase cytokine receptor complex reveals the basis for dimeric activation.

Cytokines signal through cell surface receptor dimers to initiate activation of intracellular Janus kinases (JAKs). We report the 3.6-angstrom-resolution cryo-electron microscopy structure of full-length JAK1 complexed with a cytokine receptor intracellular domain Box1 and Box2 regions captured as an activated homodimer bearing the valine→phenylalanine (VF) mutation prevalent in myeloproliferative neoplasms. The seven domains of JAK1 form an extended structural unit, the dimerization of which is mediated by close-packing of the pseudokinase (PK) domains from the monomeric subunits. The oncogenic VF mutation lies within the core of the JAK1 PK interdimer interface, enhancing packing complementarity to facilitate ligand-independent activation. The carboxy-terminal tyrosine kinase domains are poised for transactivation and to phosphorylate the receptor STAT (signal transducer and activator of transcription)-recruiting motifs projecting from the overhanging FERM (four-point-one, ezrin, radixin, moesin)-SH2 (Src homology 2)-domains. Mapping of constitutively active JAK mutants supports a two-step allosteric activation mechanism and reveals opportunities for selective therapeutic targeting of oncogenic JAK signaling.

Structural mechanism of allosteric activation of TRPML1 by PI(3,5)P2 and rapamycin

Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-permeable, nonselective cation channel ubiquitously expressed in the endolysosomes of mammalian cells and its loss-of-function mutations are the direct cause of type IV mucolipidosis (MLIV), an autosomal recessive lysosomal storage disease. TRPML1 is a ligand-gated channel that can be activated by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] as well as some synthetic small-molecule agonists. Recently, rapamycin has also been shown to directly bind and activate TRPML1. Interestingly, both PI(3,5)P2 and rapamycin have low efficacy in channel activation individually but together they work cooperatively and activate the channel with high potency. To reveal the structural basis underlying the synergistic activation of TRPML1 by PI(3,5)P2 and rapamycin, we determined the high-resolution cryoelectron microscopy (cryo-EM) structures of the mouse TRPML1 channel in various states, including apo closed, PI(3,5)P2-bound closed, and PI(3,5)P2/temsirolimus (a rapamycin analog)-bound open states. These structures, combined with electrophysiology, elucidate the molecular details of ligand binding and provide structural insight into how the TRPML1 channel integrates two distantly bound ligand stimuli and facilitates channel opening.

Cryo-EM structures of the near full-length HER2/HER3 heterodimer reveal a novel allosteric mechanism of activation

The human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) form a potent pro-oncogenic signaling unit upon binding to the extracellular ligand neuregulin-1ß (NRG1ß). Through single particle cryo-electron microscopy (cryo-EM) on graphene oxide (GO) supported grids, we have solved three novel structures of the HER2/HER3 heterodimer in the context of near-full length receptors. These structures represent the first depictions of a singly-liganded HER receptor dimer, of HER2 actively engaged with a co-receptor, and of HER3 in a ligand-bound configuration.

Structure of the human ATM kinase and mechanism of Nbs1 binding.

DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and cancer. Central to the sensing of DSBs is the ATM (Ataxia-telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family. In response to DSBs, ATM is activated by the MRN (Mre11-Rad50-Nbs1) protein complex through a poorly understood process that also requires double-stranded DNA. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM, and is required to retain active ATM at sites of DNA damage. Here, we report the 2.5 Å resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning of the N and C lobes along with relieving the blockage of the substrate-binding site. We also show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these structural findings with mutagenesis and biochemical assays.

Membrane phosphatidylserine allosterically regulates the cytosolic phospholipase A2 activity via an electrostatic-switch mechanism.

Phospholipase A2 (PLA2) is a peripheral membrane protein that plays an essential role in many inflammatory responses. However, the activation mechanisms of PLA2 on the membrane surface have not been fully understood. Herein, we have combined experimental techniques and theoretical approaches to investigate the activation and association of the PLA2 protein on an artificial phospholipid membrane. Using a phosphatidylserine (PS) nanodomain containing membrane to mimic the inflammatory conditions, we found that the activity of cytosolic PLA2s (cPLA2s) increases with higher ratios of PS in the membrane. Molecular dynamics simulations reveal that significant changes in the protein structure are related to negatively charged membranes. In particular, the alteration of negatively charged residues in the C2 domain brings about an opened binding pocket and the catalytic site access to the substrate phospholipid. Meanwhile, the negative residues in the loop 650-665 facilitate the optimal interfacial orientation of the protein with a closed binding pocket on the membrane surface. These results lead us to suggest an electrostatic-switch allosteric mechanism for cPLA2 activation on the cell membrane surface under the inflammatory state.

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss.

SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might activate SARM1. Here, we show the nicotinamide analog 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN, which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration, and neuronal death. In mice, systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to the peripheral nerve induces SARM1-dependent axon degeneration. We identify 2-aminopyridine as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity and suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

Molecular basis for the allosteric activation mechanism of the heterodimeric imidazole glycerol phosphate synthase complex

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism. HisFH is responsible for the HisH-catalyzed hydrolysis of glutamine to glutamate and ammonia, which is then used for a cyclase reaction by HisF. The HisFH complex is allosterically regulated but the underlying mechanism is not well understood. Here, we elucidate the molecular basis of the long range, allosteric activation of HisFH. We establish that the catalytically active HisFH conformation is only formed when the substrates of both HisH and HisF are bound. We show that in this conformation an oxyanion hole in the HisH active site is established, which rationalizes the observed 4500-fold allosteric activation compared to the inactive conformation. In solution, the inactive and active conformations are in a dynamic equilibrium and the HisFH turnover rates correlate with the population of the active conformation, which is in accordance with the ensemble model of allostery.

Screenning Bioactive Compounds from Allium sativum as HER2 Inhibitors Targeting Breast Cancer by Docking Methods

Screenning Bioactive Compounds from Allium sativum as HER2 Inhibitors Targeting Breast Cancer by Docking Methods

A novel RNA pol II CTD interaction site on the mRNA capping enzyme is essential for its allosteric activation.

Recruitment of the mRNA capping enzyme (CE/RNGTT) to the site of transcription is essential for the formation of the 5′ mRNA cap, which in turn ensures efficient transcription, splicing, polyadenylation, nuclear export and translation of mRNA in eukaryotic cells. The CE GTase is recruited and activated by the Serine-5 phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. Through the use of molecular dynamics simulations and enhanced sampling techniques, we provide a systematic and detailed characterization of the human CE–CTD interface, describing the effect of the CTD phosphorylation state, length and orientation on this interaction. Our computational analyses identify novel CTD interaction sites on the human CE GTase surface and quantify their relative contributions to CTD binding. We also identify, for the first time, allosteric connections between the CE GTase active site and the CTD binding sites, allowing us to propose a mechanism for allosteric activation. Through binding and activity assays we validate the novel CTD binding sites and show that the CDS2 site is essential for CE GTase activity stimulation. Comparison of the novel sites with cocrystal structures of the CE–CTD complex in different eukaryotic taxa reveals that this interface is considerably more conserved than previous structures have indicated.

Single-Molecule Insights into Allosteric Regulation of the Nuclear Receptor Protein RXRα

Nuclear receptors are crucial transcription factors that respond to a broad range of external signaling ligands by binding cognate DNA response elements and switching on transcription. As such, these proteins form tightly regulated, multi-component allosteric systems. RXRα is one of the key players in this protein family for its role as a promiscuous heterodimerization partner. We employ single-molecule fluorescence techniques to investigate the conformational dynamics of RXRα's ligand binding domain (LBD)—the essential regulatory domain. It has been proposed that ligand binding changes the conformation of helix 12 of the LBD and positions it in a conformation favoring coactivator peptide binding. Our single-molecule FRET studies, however, demonstrate that helix 12 undergoes a substantial conformational change only after the coactivator peptide binds. This suggests an unexpected allosteric mechanism of nuclear receptor activation, which we investigate. In particular, we address the questions: on what time scales do the conformational changes of helix 12 take place? and how are its dynamics affected by modulations in other parts of RXRα and in its binding partners? Our studies will pave the way to better understanding of the complex regulatory machinery embodied in nuclear receptor function.

Allosteric activation of proto-oncogene kinase Src by GPCR–beta-arrestin complexes

G protein–coupled receptors (GPCRs) initiate signaling cascades via G-proteins and beta-arrestins (βarr). βarr-dependent actions begin with recruitment of βarr to the phosphorylated receptor tail and are followed by engagement with the receptor core. βarrs are known to act as adaptor proteins binding receptors and various effectors, but it is unclear whether in addition to the scaffolding role βarrs can allosterically activate their downstream targets. Here we demonstrate the direct allosteric activation of proto-oncogene kinase Src by GPCR–βarr complexes in vitro and establish the conformational basis of the activation. Whereas free βarr1 had no effect on Src activity, βarr1 in complex with M2 muscarinic or β2-adrenergic receptors reconstituted in lipid nanodiscs activate Src by reducing the lag phase in Src autophosphorylation. Interestingly, receptor–βarr1 complexes formed with a βarr1 mutant, in which the finger-loop, required to interact with the receptor core, has been deleted, fully retain the ability to activate Src. Similarly, βarr1 in complex with only a phosphorylated C-terminal tail of the vasopressin 2 receptor activates Src as efficiently as GPCR–βarr complexes. In contrast, βarr1 and chimeric M2 receptor with nonphosphorylated C-terminal tail failed to activate Src. Taken together, these data demonstrate that the phosphorylated GPCR tail interaction with βarr1 is necessary and sufficient to empower it to allosterically activate Src. Our findings may have implications for understanding more broadly the mechanisms of allosteric activation of downstream targets by βarrs.

The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.

Mechanism of allosteric activation of SIRT6 revealed by the action of rationally designed activators

The recent discovery of activator compounds binding to an allosteric site on the NAD+-dependent protein lysine deacetylase, sirtuin 6 (SIRT6) has attracted interest and presents a pharmaceutical target for aging-related and cancer diseases. However, the mechanism underlying allosteric activation of SIRT6 by the activator MDL-801 remains largely elusive because no major conformational changes are observed upon activator binding. By combining molecular dynamics simulations with biochemical and kinetic analyses of wild-type SIRT6 and its variant M136A, we show that conformational rotation of 2-methyl-4-fluoro-5-bromo substituent on the right phenyl ring (R-ring) of MDL-801, which uncovers previously unseen hydrophobic interactions, contributes to increased activating deacetylation activity of SIRT6. This hypothesis is further supported by the two newly synthesized MDL-801 derivatives through the removal of the 5-Br atom on the R-ring (MDL-801-D1) or the restraint of the rotation of the R-ring (MDL-801-D2). We further propose that the 5-Br atom serves as an allosteric driver that controls the ligand allosteric efficacy. Our study highlights the effect of allosteric enzyme catalytic activity by activator binding and provides a rational approach for enhancing deacetylation activity.

Orthosteric and Allosteric Activation of Human 5-HT3A Receptors

The serotonin type 3 receptor (5-HT3) is a ligand-gated ion channel that converts the binding of the neurotransmitter serotonin (5-HT) into a transient cation current that mediates fast excitatory responses in peripheral and central nervous systems. Information regarding the activation and modulation of the human 5-HT3 type A receptor has been based only on macroscopic current measurements because of its low ion conductance. By constructing a high-conductance human 5-HT3A receptor, we here revealed mechanistic information regarding the orthosteric activation by 5-HT and by the partial agonist tryptamine, and the allosteric activation by the terpenoids, carvacrol, and thymol. Terpenoids potentiated macroscopic currents elicited by the orthosteric agonist and directly elicited currents with slow-rising phases and submaximal amplitudes. At the single-channel level, activation by orthosteric and allosteric agonists appeared as openings in quick succession (bursts) that showed no ligand concentration dependence. Bursts were grouped into long-duration clusters in the presence of 5-HT and even longer in the presence of terpenoids, whereas they remained isolated in the presence of tryptamine. Kinetic analysis revealed that allosteric and orthosteric activation mechanisms can be described by the same scheme that includes transitions of the agonist-bound receptor to closed intermediate states before opening (priming). Reduced priming explained the partial agonism of tryptamine; however, equilibrium constants for gating and priming were similar for 5-HT and terpenoid activation. Thus, our kinetic analysis revealed that terpenoids are efficacious agonists for 5-HT3A receptors. These findings not only extend our knowledge about the human 5-HT3A molecular function but also provide novel insights into the mechanisms of action of allosteric ligands, which are of increasing interest as therapeutic drugs in all the superfamily.

Catalytic Domain Plasticity of MKK7 Reveals Structural Mechanisms of Allosteric Activation and Diverse Targeting Opportunities

MKK7 (MEK7) is a key regulator of the JNK stress signaling pathway and targeting MKK7 has been proposed as a chemotherapeutic strategy. Detailed understanding of the MKK7 structure and factors that affect its activity is therefore of critical importance. Here, we present a comprehensive set of MKK7 crystal structures revealing insights into catalytic domain plasticity and the role of the N-terminal regulatory helix, conserved in all MAP2Ks, mediating kinase activation. Crystal structures harboring this regulatory helix revealed typical structural features of active kinase, providing exclusively a first model of the MAP2K active state. A small-molecule screening campaign yielded multiple scaffolds, including type II irreversible inhibitors a binding mode that has not been reported previously. We also observed an unprecedented allosteric pocket located in the N-terminal lobe for the approved drug ibrutinib. Collectively, our structural and functional data expand and provide alternative targeting strategies for this important MAP2K kinase.

Insights into ADAMTS13 structure: impact on thrombotic thrombocytopenic purpura diagnosis and management.

Fundamental knowledge on the role of a disintegrin and metalloprotease with thrombospondin type one repeats, member 13 (ADAMTS13) has been crucial to better understand the pathophysiology of the rare and life-threatening disease thrombotic thrombocytopenic purpura (TTP). ADAMTS13 works through a molecular zipper mechanism to proteolyze its substrate von Willebrand factor (VWF). Recent insights into the structure and function of ADAMTS13 led to the identification of an allosteric activation mechanism. Therefore, ADAMTS13 is roughly folded in two in which the N-terminal spacer (S) domain and C-terminal T7-CUB2 domains interact to adopt a closed conformation. Upon substrate binding, ADAMTS13 adopts an open conformation in which the S-T7-CUB2 interaction is abrogated to further position VWF towards the catalytic cleft, inducing activation of the latent metalloprotease domain and resulting in cleavage of VWF. Unravelling the structure function relationship of ADAMTS13 helped identifying open ADAMTS13 as a novel and unique biomarker for immune-mediated TTP (iTTP). This novel biomarker has potential in the diagnosis, treatment and follow-up of iTTP. In this review, the most recent findings on the structure and working mechanism of ADAMTS13 are addressed. In addition, how those findings led to the identification of a novel biomarker, and how this novel biomarker could have an impact on the diagnosis, management and follow-up of iTTP patients are discussed.

Different Solvent and Conformational Entropy Contributions to the Allosteric Activation and Inhibition Mechanisms of Yeast Chorismate Mutase.

Allosteric regulation is important in many biological processes, including cell signaling, gene regulation, and metabolism. Saccharomyces cerevisiae chorismate mutase (ScCM) is a key homodimeric enzyme in the shikimate pathway responsible for the generation of aromatic amino acids, where it is allosterically inhibited and activated by Tyr and Trp, respectively. Our previous studies indicated that binding of both allosteric effectors is negatively cooperative, that is binding at one allosteric binding site discourages binding at the other, due to the entropic penalty of binding the second allosteric effector. We utilized variable temperature isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments to better understand the entropic contributions to allosteric effector binding, including changes to solvent entropy and protein conformational entropy. Upon binding either Tyr or Trp, ScCM experiences a quenching of motions on the picosecond-to-nanosecond time scale, which we could relate to a loss of protein conformational entropy. Further ITC and NMR studies were consistent with the Tyr-bound form of ScCM being associated with more water molecules compared to the Trp-bound form and Tyr binding being associated with a less positive solvent entropy change. These studies provide insight into the role of structural dynamics in ScCM function and highlight the importance of solvent entropy changes in allosteric regulation, a historically underappreciated concept.