Allosteric Switch Research Articles

Helix-Based RNA Landscape Partition and Alternative Secondary Structure Determination.

RNA is a versatile macromolecule with the ability to fold into and interconvert between multiple functional conformations. The elucidation of the RNA folding landscape, especially the knowledge of alternative structures, is critical to uncover the physical mechanism of RNA functions. Here, we introduce a helix-based strategy for RNA folding landscape partition and alternative secondary structure determination. The benchmark test of 27 RNAs involving alternative stable structures shows that the model has the ability to divide the whole landscape into distinct partitions at the secondary structure level and predict the representative structures for each partition. Furthermore, the predicted structures and equilibrium populations of metastable conformations for the 2′dG-sensing riboswitch reveal the allosteric conformational switch on transcript length, which is consistent with the experimental study, indicating the importance of metastable states for RNA-based gene regulation. The model delivers a starting point for the landscape-based strategy toward the RNA folding mechanism and functions.

Co-temporal Force and Fluorescence Measurements Reveal a Ribosomal Gear Shift Mechanism of Translation Regulation by Structured mRNAs.

The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.

NMR structure determination of Ixolaris and factor X(a) interaction reveals a noncanonical mechanism of Kunitz inhibition

AbstractIxolaris is a potent tick salivary anticoagulant that binds coagulation factor Xa (FXa) and zymogen FX, with formation of a quaternary tissue factor (TF)/FVIIa/ FX(a)/Ixolaris inhibitory complex. Ixolaris blocks TF-induced coagulation and PAR2 signaling and prevents thrombosis, tumor growth, and immune activation. We present a high-resolution structure and dynamics of Ixolaris and describe the structural basis for recognition of FX. Ixolaris consists of 2 Kunitz domains (K1 and K2) in which K2 is strikingly dynamic and encompasses several residues involved in FX binding. This indicates that the backbone plasticity of K2 is critical for Ixolaris biological activity. Notably, a nuclear magnetic resonance–derived model reveals a mechanism for an electrostatically guided, high-affinity interaction between Ixolaris and FX heparin-binding (pro)exosite, resulting in an allosteric switch in the catalytic site. This is the first report revealing the structure-function relationship of an anticoagulant targeting a zymogen serving as a scaffold for TF inhibition.

A label-free IFN-γ aptasensor based on target-triggered allosteric switching of aptamer beacon and streptavidin-inorganic hybrid composites

A label-free electrochemical aptasensor was developed for the sensitive detection of interferon-gamma (IFN-γ). To do this, a diblock dual-aptamer allosteric hairpin (DDAH) was designed, followed by conjugation with gold nanoparticles (DDAH&AuNP). The presence of target destroyed the stable hairpin structure, and then the catalytic cleavage of DNAzymes removed the IFN-γ-binding molecules, triggering the allosteric switching from inactive hairpin to active streptavidin aptamer (A-DDAH&AuNP) in homogeneous system. Moreover, streptavidin-inorganic hybrid nanoflowers decorated with graphene composites (SFG) were synthesized and used as substrates to modify glassy carbon electrodes (SFG/GCE). SFG specifically bind to the A-DDAH&AuNP to realize high-efficient readout of signals. Under the optimal conditions and by using differential pulse stripping voltammetry (DPSV), the response peak currents increases linearly with the logarithm of the IFN-γ concentration in the range between 0.1 pg mL−1 and 500 ng/mL. The detection limit is as low as 19 fg mL−1. The aptasensor also has excellent electrochemical performances, which exhibits broad application prospects in biometric analysis.

A switch element in the autophagy E2 Atg3 mediates allosteric regulation across the lipidation cascade

Autophagy depends on the E2 enzyme, Atg3, functioning in a conserved E1-E2-E3 trienzyme cascade that catalyzes lipidation of Atg8-family ubiquitin-like proteins (UBLs). Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12–Atg5-Atg16 with pathologies including cancers, infections and neurodegeneration. Here, studying yeast enzymes, we report that an Atg3 element we term E123IR (E1, E2, and E3-interacting region) is an allosteric switch. NMR, biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3E123IR makes intramolecular interactions restraining Atg3′s catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3′s E123IR protects the E2~UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation cascade enzymes induce E2 active site remodeling through an unprecedented mechanism to drive autophagy.

Apoptosis-inducing factor: mechanistic insights and therapeutic opportunities from a metabolic allosteric switch

Apoptosis-inducing factor: mechanistic insights and therapeutic opportunities from a metabolic allosteric switch

Generalizable Protein Biosensors Based on Synthetic Switch Modules.

Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor's activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.

A role for allosteric disulfides in regulating the function of human killer cell receptor KIR2DL4

Abstract KIR2DL4 is an unusual member of the killer cell Ig-like receptor (KIR) family expressed in NK cells. It resides mostly in endosomes and activates NK cells in response to soluble agonists, such as soluble HLA-G and a monoclonal antibody. KIR2DL4 signals from endosomes for a unique proinflammatory and proangiogenic response, which has relevance to placental development due to the restricted expression of its ligand, HLA-G, by fetal trophoblast cells during early pregnancy. To understand the nature and regulation of KIR2DL4–HLA-G interactions, we used an unbiased random mutagenesis approach and screened mutants of KIR2DL4 in a cellular assay for their ability to load soluble HLA-G into KIR2DL4+ endosomes. With this approach, amino acid residues in KIR2DL4 that are important for endosomal localization and HLA-G recognition were identified. Specifically, we have identified a putative allosteric cysteine in the extracellular domain of this receptor, which may be involved in a disulfide switching mechanism to control receptor internalization and ligand binding. Protein disulfide isomerase (PDI) acting at the cell surface may be responsible for this switch as inhibition of PDI interfered with internalization of KIR2DL4 and uptake of HLA-G into endosomes. The configuration of the relevant disulfide bond is -RHstaple, which is a hallmark of many allosteric disulfides. Thus, KIR2DL4 localization and function is regulated by allosteric disulfide bonds at the cell surface. Studies are underway to explore the dynamic regulation of receptor structure by this allosteric disulfide switch to control ligand binding, localization and signaling by this receptor.

The juxtamembrane linker in neutral sphingomyelinase-2 functions as an intramolecular allosteric switch that activates the enzyme

Neutral sphingomyelinase 2 (nSMase2) produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation. Although nSMase2 has low basal activity, it is fully activated by phosphatidylserine (PS). Previous work showed that interdomain interactions within nSMase2 are needed for PS activation. Here, we use multiple approaches, including small angle X-ray scattering, hydrogen-deuterium exchange-MS, circular dichroism and thermal shift assays, and membrane yeast two-hybrid assays, to define the mechanism mediating this interdomain interactions within nSMase2. In contrast to what we previously assumed, we demonstrate that PS binding at the N-terminal and juxtamembrane regions of nSMase2 rather acts as a conformational switch leading to interdomain interactions that are critical to enzyme activation. Our work assigns a unique function for a class of linkers of lipid-activated, membrane-associated proteins. It indicates that the linker actively participates in the activation mechanism via intramolecular interactions, unlike the canonical linkers that typically aid protein dimerization or localization.

(p)ppGpp Regulates a Bacterial Nucleosidase by an Allosteric Two-Domain Switch.

The stringent response alarmones pppGpp and ppGpp are essential for rapid adaption of bacterial physiology to changes in the environment. In Escherichia coli, the nucleosidase PpnN (YgdH) regulates purine homeostasis by cleaving nucleoside monophosphates and specifically binds (p)ppGpp. Here, we show that (p)ppGpp stimulates the catalytic activity of PpnN both invitro and invivo causing accumulation of several types of nucleobases during stress. The structure of PpnN reveals a tetramer with allosteric (p)ppGpp binding sites located between subunits. pppGpp binding triggers a large conformational change that shifts the two terminal domains to expose the active site, providing a structural rationale for the stimulatory effect. We find that PpnN increases fitness and adjusts cellular tolerance to antibiotics and propose a model in which nucleotide levels can rapidly be adjusted during stress by simultaneous inhibition of biosynthesis and stimulation of degradation, thus achieving a balanced physiological response to constantly changing environments.

Identifying an Allosteric Switch Region in Human SIRT1

SIRT1 is an NAD+‐dependent lysine deacetylase that is implicated in age‐related diseases and is a potential therapeutic target for drug development. The N‐terminal domain is highly disordered and is usually removed prior to crystallization of the enzyme. However, it has been known to physically interact with the catalytic core to increase enzyme activity. The mechanism of this remains unknown. STAC binding domain (SBD), within the N‐terminal region, is the binding site for sirtuin‐activating compounds (STACs) such as resveratrol, which is known to enhance SIRT1 activity towards peptide substrates including p53W. An allosteric switch is hypothesized as a single or multiple residues responsible for a change in secondary structure essential for allosteric activity and mostly found in disordered regions. Detailed information on switch regions remains unexplored due to the challenges of crystallizing the disordered regions. Our project aims to identify and confirm an allosteric switch region within the N‐terminal domain of SIRT1. The first aim is to computationally identify a switch region in SIRT1 using sequence‐and homology‐based descriptors. A possible switch region is hypothesized as having (1) high secondary structure variability, (2) low sequence entropy, and (3) low sequence disorder propensity, inferred by a combination of predictors. Applying these predictors to the full sequence of SIRT1 showed that such a switch may occur within residues 186–193 (TFVQQHLM) of the SBD. The second aim is to experimentally confirm these predictions. We have mutated the predicted region to serine residues: 186–189S, 190–193S, 188S, and 192S. The enzyme kinetics of the mutants remain relatively similar to the wild‐type SIRT1. We then tested these mutants for susceptibility to allosteric modulations by assaying their enzyme activity towards p53W with and without resveratrol. No increase in activity with the addition of resveratrol would confirm that these residues are involved in an allosteric switch. These results will inform the accuracy of the computational descriptors and provide a better understanding of the regulation of this enzyme in the cellular system.Support or Funding InformationCalifornia State University Program for Education and Research in Biotechnology; Louis Stokes Alliance for Minority ParticipationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Detection of beta1 integrin activation in chondrocytes

Purpose: Integrins are heterodimeric transmembrane receptors linking the actin cytoskeleton to the ECM. Their activity is regulated via an allosteric switch between low- and high-affinity states, induced by effector binding to the cytoplasmic tail of the α- and β-subunits or by ligand binding to the ectodomain. β1 integrin activity in chondrocytes has been shown to increase during osteoarthritis leading to increased catabolism, however β1 integrin signalling is required during chondrogenic differentiation. Integrin signalling is involved in mechanisms of cell survival, growth, differentiation and matrix remodelling, however the exact mechanisms by which β1 integrin signalling may interact with other homeostatic signalling pathways in chondrocytes remains unknown. Here, we aim to examine the expression pattern of β1 integrins within their activated state in chondrocytes. Methods: Costal chondrocytes obtained from 8 to 12-week-old male wild type (WT) mice were cultured under standard conditions. β1 integrin within an active conformation was detected by immunofluorescence staining using a Purified Rat Anti-Mouse CD29 Clone 9EG7 antibody. Activation levels were compared in chondrocytes plated for 24 hours on either fibronectin, collagen type II or uncoated glass coverslips. Chondrocytes in non-coated wells were stimulated in a time course with 1 mM Mn2+ to act as a positive control. To further investigate β1 integrin activation as part of attachment behaviour, chondrocytes were added to fibronectin coated and uncoated coverslips for 1 hour, 6 hours and 24 hours before β1 integrin activation was assessed by immunocytochemistry. Integrin-specific downstream signalling was analysed by immunoblotting of p-FAK/FAK and PKC in lysates of treated chondrocytes. Results: β1 integrins within an active conformation could be detected in wild type murine chondrocytes in vitro. The 9EG7 epitope was detected firstly at low levels in chondrocytes grown in standard conditions, however activation levels were found to increase significantly when chondrocytes were cultured on either fibronectin or type II collagen coatings. Mn2+ added to chondrocytes cultured in uncoated wells as a positive control led to increased levels of activated β1 integrin. Activated integrin β1 in chondrocytes grown on fibronectin and type II collagen exhibited a specific activation pattern, which was not found in chondrocytes grown on uncoated surfaces. Immunoblotting demonstrated that activation of β1 integrins via manganese or through interaction with RGD sequence containing ECM substrates led to increased phosphorylation of the focal adhesion kinase and protein kinase C. Conclusions: β1 integrins within an active conformation were able to be detected in in vitro cultured chondrocytes using immunocytochemistry for the 9EG7 epitope of β1 integrin. The activated state was increased both by Mn2+ and when chondrocytes were grown on either fibronectin or type II collagen substrates. This detection of β1 integrin activation state allows for the further investigation of integrin signalling within the context of interactions with homeostatic growth factor and chemokine signalling pathways.

Tunable microsecond dynamics of an allosteric switch regulate the activity of a AAA+ disaggregation machine

Large protein machines are tightly regulated through allosteric communication channels. Here we demonstrate the involvement of ultrafast conformational dynamics in allosteric regulation of ClpB, a hexameric AAA+ machine that rescues aggregated proteins. Each subunit of ClpB contains a unique coiled-coil structure, the middle domain (M domain), proposed as a control element that binds the co-chaperone DnaK. Using single-molecule FRET spectroscopy, we probe the M domain during the chaperone cycle and find it to jump on the microsecond time scale between two states, whose structures are determined. The M-domain jumps are much faster than the overall activity of ClpB, making it an effectively continuous, tunable switch. Indeed, a series of allosteric interactions are found to modulate the dynamics, including binding of nucleotides, DnaK and protein substrates. This mode of dynamic control enables fast cellular adaptation and may be a general mechanism for the regulation of cellular machineries.

Emerging paradigms for PilZ domain-mediated C-di-GMP signaling

PilZ domain-containing proteins constitute a large family of bacterial signaling proteins. As a widely distributed protein domain for the binding of the second messenger c-di-GMP, the canonical PilZ domain contains a set of motifs that define the binding site for c-di-GMP and an allosteric switch for propagating local conformational changes. Here, we summarize some new insights gathered from recent studies on the commonly occurring single-domain PilZ proteins, YcgR-like proteins and PilZ domain-containing cellulose synthases. The studies collectively illuminate how PilZ domains function as cis- or trans-regulatory domains that enable c-di-GMP to control the activity of its cellular targets. Overall, the review highlights the diverse protein structure, biological function and regulatory mechanism of PilZ domain-containing proteins, as well as the challenge of deciphering the function and mechanism of orphan PilZ proteins.

Bioelectrocatalytic Electrodes Modified with PQQ‐Glucose Dehydrogenase‐Calmodulin Chimera Switchable by Peptide Signals: Pathway to Generic Bioelectronic Systems Controlled by Biomolecular Inputs

AbstractConstruction of artificial allosteric protein switches is one of the central goals of synthetic biology that holds promise to transform the way we detect and quantify substances in vitro and in vivo. An artificial chimeric fusion protein of pyrroloquinoline quinone‐dependent glucose dehydrogenase with calmodulin (PQQ‐GDH‐CaM) was covalently attached to graphene nanosheets produced electrochemically on a carbon fiber electrode. The chimeric PQQ‐GDH‐CaM represents an artificial allosteric switch activated by association of a calmodulin‐binding peptide with the Ca2+‐bound calmodulin domain. The activity of the immobilized enzyme was switched between active and inactive states by adding/removing the activating peptide. The peptide‐signal switchable features originated from the enzyme 3D‐structural variations induced by the conformational (folding/unfolding) changes in the connected calmodulin unit upon formation/dissociation of its complex with the specific peptide. The peptide‐activated immobilized PQQ‐GDH‐CaM enzyme displayed direct (non‐mediated) electron transfer to the conducting electrode support upon glucose oxidation. On the contrary, in the absence of the peptide, the inactive form of the enzyme demonstrated very low bioelectrocatalytic activity for glucose oxidation. Since the conformational changes of the PQQ‐GDH‐CaM depend on the presence of Ca2+ cations and the calmodulin‐binding peptide, both of them were used as input signals to control the enzyme activity mimicking a Boolean AND logic gate. The switchable behavior of the enzyme‐modified electrode was studied electrochemically and used to assemble a signal‐switchable biofuel cell. The use of the peptide as the signaling messenger enables the design of generalizable bioelectronic systems controlled by native and synthetic biochemical signaling systems.

Metalloprotein switches that display chemical-dependent electron transfer in cells.

Biological electron transfer is challenging to directly regulate using environmental conditions. To enable dynamic, protein-level control over energy flow in metabolic systems for synthetic biology and bioelectronics, we created ferredoxin logic gates that utilize transcriptional and post-translational inputs to control energy flow through a synthetic electron transfer pathway that is required for bacterial growth. These logic gates were created by subjecting a thermostable, plant-type ferredoxin to backbone fission and fusing the resulting fragments to a pair of proteins that self-associate, a pair of proteins whose association is stabilized by a small molecule, and to the termini of a ligand-binding domain. We show that the latter domain insertion design strategy yields an allosteric ferredoxin switch that acquires an oxygen-tolerant [2Fe-2S] cluster and can use different chemicals, including a therapeutic drug and an environmental pollutant, to control the production of a reduced metabolite in Escherichia coli and cell lysates.

DNA-Walker-Induced Allosteric Switch for Tandem Signal Amplification with Palladium Nanoparticles/Metal-Organic Framework Tags in Electrochemical Biosensing.

A DNA walker as a new molecular machine can walk on defined tracks to directly generate signal indicators in biosensing and biomedical applications. In this work, a tandem signal amplification strategy was developed on the basis of the DNA-walker-induced conformation switch for bridging palladium nanoparticles/metal-organic framework tags in ultrasensitive electrochemical DNA biosensing. The signal tags were synthesized by in situ reduction of Pd nanocrystals on porphyrinic metal-organic frameworks (PCN-224), followed by conjugation with streptavidin (SA). The as-prepared Pd/PCN-224-SA tag could electrocatalyze the oxidation of NaBH4 with high efficiency for signal readout. The presence of target DNA released swing arms that were each silenced by a blocker, and then the activated swing arms could hybridize with hairpin DNA. The movement of swing arms was powered by enzymatic cleavage of conjugated oligonucleotides, inducing the allosteric switch from hairpin to SA aptamer. Therefore, Pd/PCN-224-SA tags were brought onto the electrode surface via SA-aptamer biorecognition to generate the enhanced electrochemical signal. The DNA walker-based electrochemical biosensor demonstrated good performance such as 6 orders of magnitude linear range, femtomolar detection limit, and single mismatch differentiation ability. Moreover, the feasibility of the biosensor was identified in serum matrixes. The tandem signal amplification of metal-organic frameworks and DNA walkers provided a new avenue in trace electrochemical biosensing.

Hysteresis and Allostery in Human UDP-Glucose Dehydrogenase Require a Flexible Protein Core

Human UDP-glucose dehydrogenase (hUGDH) oxidizes UDP-glucose to UDP-glucuronic acid, an essential substrate in the phase II metabolism of drugs. The activity of hUGDH is regulated by the conformation of a buried allosteric switch (T131 loop/α6 helix). Substrate binding induces the allosteric switch to slowly isomerize from an inactive E* conformation to the active E state, which can be observed as enzyme hysteresis. When the feedback inhibitor UDP-xylose binds, the allosteric switch and surrounding residues in the protein core repack, converting the hexamer into an inactive, horseshoe-shaped complex (EΩ). This allosteric transition is facilitated by large cavities and declivities in the protein core that provide the space required to accommodate the alternate packing arrangements. Here, we have used the A104L substitution to fill a cavity in the E state and sterically prevent repacking of the core into the EΩ state. Steady state analysis shows that hUGDHA104L binds UDP-xylose with lower affinity and that the inhibition is no longer cooperative. This means that the allosteric transition to the high-UDP-xylose affinity EΩ state is blocked by the substitution. The crystal structures of hUGDHA104L show that the allosteric switch still adopts the E and E* states, albeit with a more rigid protein core. However, the progress curves of hUGDHA104L do not show hysteresis, which suggests that the E* and E states are now in rapid equilibrium. Our data suggest that hysteresis in native hUGDH originates from the conformational entropy of the E* state protein core.

Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism.

Protein kinases achieve substrate selective phosphorylation through their conformational flexibility and dynamic interaction with the substrate. Designing substrate selective or kinase selective small molecule inhibitors remains a challenge because of a lack of understanding of the dynamic mechanism by which substrates are selected by the kinase. Using a combination of all-atom molecular dynamics simulations and FRET sensors, we have delineated an allosteric mechanism that results in interaction among the DFG motif, G-loop, and activation loop and structurally links the nucleotide and substrate binding interfaces in protein kinase Cα and three other Ser/Thr kinases. ATP-competitive staurosporine analogues engage this allosteric switch region located just outside the ATP binding site to displace substrate binding to varying degrees. These inhibitors function as bitopic ligands by occupying the ATP binding site and interacting with the allosteric switch region. The conserved mechanism identified in this study can be exploited to select and design bitopic inhibitors for kinases.

Positive/Negative Allosteric Modulation Switching in an Umami Taste Receptor (T1R1/T1R3) by a Natural Flavor Compound, Methional

Taste is a vital sensation for vertebrates, enabling the detection of nutritionally important substances or potential toxins. A heteromeric complex of two class C GPCRs, T1R1 and T1R3, was identified as the umami (savory) taste receptor. Amino acids and 5′-ribonucleotides are well known to be natural ligands for human T1R1/T1R3. In this study, we reveal that methional, which is a familiar flavor component in foods, is an allosteric modulator of T1R1/T1R3. Receptor expression experiments showed that methional served as a positive allosteric modulator (PAM) of human T1R1/T1R3 and functioned as a negative allosteric modulator (NAM) of mouse T1R1/T1R3. Although amino acids and 5′-ribonucleotides bound to the extracellular domain of T1R1, the use of interspecies chimeric receptors demonstrated that methional interacted with the transmembrane domain of T1R1. Site-directed mutagenesis and molecular modeling showed that methional could potentially bind at two distinct sites in the transmembrane domain of T1R1 and that the amino acid residues in the bottom of the allosteric pocket engendered the switch between the PAM and NAM modes, which could contribute to switching the binding position of methional. These results may be applicable for elucidating the molecular mechanisms underlying ligand recognition by other class C GPCRs.