Conformational Selection Mechanism Research Articles

Real-time single-molecule observation of chaperone-assisted protein folding.

The ability of heat shock protein 70 (Hsp70) molecular chaperones to remodel the conformation of their clients is central to their biological function; however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes toward efficient protein folding. Here, we used total internal reflection fluorescence (TIRF) microscopy and single-molecule fluorescence resonance energy transfer (smFRET) to temporally observe the conformational changes that occur to individual firefly luciferase proteins as they are folded by the bacterial Hsp70 system. We observed multiple cycles of chaperone binding and release to an individual client during refolding and determined that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism, whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.

Mixed mechanism of conformational selection and induced fit as a molecular recognition process in the calreticulin family of proteins.

The fundamental question on the mechanism of molecular recognition during ligand binding has attracted a lot of scientific scrutiny. The two competing theories of ligand binding-"induced fit" and "conformational selection" have been proposed to explain biomolecular recognition. Since exploring a family of proteins with similar structural architectures and conserved functional roles can provide valuable insight into the significance of molecular structure and function, we performed molecular dynamics simulations on the calreticulin family of proteins, which specifically recognize monoglucosylated N-glycan during the protein folding process. Atomistic simulations of lectins in free and bound forms demonstrated that they exist in several conformations spanning from favorable to unfavorable for glycan binding. Our analysis was confined to the carbohydrate recognition domain (CRD) of these lectins to demonstrate the degree of conservation in protein sequence and structure and relate them with their function. Furthermore, we computed the lectin-glycan binding affinity using the mmPBSA approach to identify the most favorable lectin conformation for glycan binding and compared the molecular interaction fields in terms of noncovalent bond interactions. We also demonstrated the involvement of Tyr and Trp residues in the CRD with the non-reducing end glucose and central mannose residues, which contribute to some of the specific interactions. Furthermore, we analyzed the conformational changes in the CRD through SASA, RMSFs and protein surface topography mapping of electrostatic and hydrophobic potentials. Our findings demonstrate a hybrid mechanism of molecular recognition, initially driven by conformational selection followed by glycan-induced fluctuations in the key residues to strengthen the glycan binding interactions.

Molecular mechanism underlying the di-uridylation activity of Arabidopsis TUTase URT1.

In Arabidopsis, HESO1 and URT1 act cooperatively on unmethylated miRNA and mRNA uridylation to induce their degradation. Their collaboration significantly impacts RNA metabolism in plants. However, the molecular mechanism determining the functional difference and complementarity of these two enzymes remains unclear. We previously solved the three-dimensional structure of URT1 in the absence and presence of UTP. In this study, we further determined the structure of URT1 in complex with a 5′-AAAU-3′ RNA stretch that mimics the post-catalytic state of the mRNA poly(A) tail after the addition of the first uridine. Structural analysis and enzymatic assays revealed that L527 and Y592 endow URT1 with a preference to interact with purine over pyrimidine at the -1 RNA binding position, thus controlling the optimal number of uridine added to the 3′ extremity of poly(A) as two. In addition, we observed that a large-scale conformational rearrangement in URT1 occurs upon binding with RNA from an ‘open’ to a ‘closed’ state. Molecular dynamic simulation supports an open-closed conformational selection mechanism employed by URT1 to interact with RNA substrates and maintain distributive enzymatic activity. Based on the above results, a model regarding the catalytic cycle of URT1 is proposed to explain its di-uridylation activity.

Structural basis of ion – substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY

The Na+-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na+ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen cooperative mechanism by conformational selection. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPinb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY’s conformational selection mechanism is a result of the sodium-dependent formation of the substrate binding site.

Using Big Data Analytics to "Back Engineer" Protein Conformational Selection Mechanisms.

In the living cells, proteins bind small molecules (or “ligands”) through a “conformational selection” mechanism, where a subset of protein structures are capable of binding the small molecules well while most other protein structures are not capable of such binding. The present work uses machine learning approaches to identify, in a very large amount of protein:ligand complexes, what protein properties are associated with their capacity to bind small molecules. In order to do so, we calculate 40 physicochemical properties on about 1.5 millions of protein conformations: ligand and protein conformations. This work describes a machine learning approach to identify the unique physico-chemical descriptors of a protein that maximize the prediction rate of potential protein molecular conformations for the test case proteins ADORA2A (Adenosine A2a Receptor), ADRB2 (Adrenoceptor Beta 2) and OPRK1 (Opioid Receptor Kappa 1). We find adequate machine learning techniques can increase by an order of magnitude the identification of “binding protein conformations” in an otherwise very large ensemble of protein conformations, compared to random selection of protein conformations. This opens the door to the systematic identification of such “binding conformations” for proteins and provides a big data approach to the conformational selection mechanism.

Early aggregation mechanism of Aβ16−22 revealed by Markov state models

Aβ16–22 is believed to have critical role in early aggregation of full length amyloids that are associated with the Alzheimer's disease and can aggregate to form amyloid fibrils. However, the early aggregation mechanism is still unsolved. Here, multiple long-term molecular dynamics simulations combining with Markov state model were used to probe the early oligomerization mechanism of Aβ16−22 peptides. The identified dimeric form adopted either globular random-coil or extended β-strand like conformations. The observed dimers of these variants shared many overall conformational characteristics but differed in several aspects at detailed level. In all cases, the most common type of secondary structure was intermolecular antiparallel β-sheets. The inter-state transitions were very frequent ranges from few to hundred nanoseconds. More strikingly, those states which contain fraction of β secondary structure and significant amount of extended coiled structures, therefore exposed to the solvent, were majorly participated in aggregation. The assembly of low-energy dimers, in which the peptides form antiparallel β sheets, occurred by multiple pathways with the formation of an obligatory intermediates. We proposed that these states might facilitate the Aβ16−22 aggregation through a significant component of the conformational selection mechanism, because they might increase the aggregates population by promoting the inter-chain hydrophobic and the hydrogen bond contacts. The formation of early stage antiparallel β sheet structures is critical for oligomerization, and at the same time provided a flat geometry to seed the ordered β-strand packing of the fibrils. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aβ aggregation and will insight into early oligomerization for large β amyloids.

Structural-Energetic Basis for Coupling between Equilibrium Fluctuations and Phosphorylation in a Protein Native Ensemble.

The functioning of proteins is intimately tied to their fluctuations in the native ensemble. The structural–energetic features that determine fluctuation amplitudes and hence the shape of the underlying landscape, which in turn determine the magnitude of the functional output, are often confounded by multiple variables. Here, we employ the FF1 domain from human p190A RhoGAP protein as a model system to uncover the molecular basis for phosphorylation of a buried tyrosine, which is crucial to the transcriptional activity associated with transcription factor TFII-I. Combining spectroscopy, calorimetry, statistical–mechanical modeling, molecular simulations, and in vitro phosphorylation assays, we show that the FF1 domain samples a diverse array of conformations in its native ensemble, some of which are phosphorylation-competent. Upon eliminating unfavorable charge–charge interactions through a single charge-reversal (K53E) or charge-neutralizing (K53Q) mutation, we observe proportionately lower phosphorylation extents due to the altered structural coupling, damped equilibrium fluctuations, and a more compact native ensemble. We thus establish a conformational selection mechanism for phosphorylation in the FF1 domain with K53 acting as a “gatekeeper”, modulating the solvent exposure of the buried tyrosine. Our work demonstrates the role of unfavorable charge–charge interactions in governing functional events through the modulation of native ensemble characteristics, a feature that could be prevalent in ordered protein domains.

Rational design of a helical peptide inhibitor targeting c-Myb\u2013KIX interaction

The transcription factor c-Myb promotes the proliferation of hematopoietic cells by interacting with the KIX domain of CREB-binding protein; however, its aberrant expression causes leukemia. Therefore, inhibitors of the c-Myb–KIX interaction are potentially useful as antitumor drugs. Since the intrinsically disordered transactivation domain (TAD) of c-Myb binds KIX via a conformational selection mechanism where helix formation precedes binding, stabilizing the helical structure of c-Myb TAD is expected to increase the KIX-binding affinity. Here, to develop an inhibitor of the c-Myb–KIX interaction, we designed mutants of the c-Myb TAD peptide fragment where the helical structure is stabilized, based on theoretical predictions using AGADIR. Three of the four initially designed peptides each had a different Lys-to-Arg substitution on the helix surface opposite the KIX-binding interface. Furthermore, the triple mutant with three Lys-to-Arg substitutions, named RRR, showed a high helical propensity and achieved three-fold higher affinity to KIX than the wild-type TAD with a dissociation constant of 80 nM. Moreover, the RRR inhibitor efficiently competed out the c-Myb–KIX interaction. These results suggest that stabilizing the helical structure based on theoretical predictions, especially by conservative Lys-to-Arg substitutions, is a simple and useful strategy for designing helical peptide inhibitors of protein–protein interactions.

The active site region plays a critical role in Na+ binding to thrombin

The catalytic activity of thrombin and other enzymes of the blood coagulation and complement cascades is enhanced significantly by binding of Na+ to a site >15 Å away from the catalytic residue S195, buried within the 180 and 220 loops that also contribute to the primary specificity of the enzyme. Rapid kinetics support a binding mechanism of conformational selection where the Na+-binding site is in equilibrium between open (N) and closed (N∗) forms and the cation binds selectively to the N form. Allosteric transduction of this binding step produces enhanced catalytic activity. Molecular details on how Na+ gains access to this site and communicates allosterically with the active site remain poorly defined. In this study, we show that the rate of the transition is strongly correlated with the analogous transition that governs the interaction of synthetic and physiologic substrates with the active site. This correlation supports the active site as the likely point of entry for Na+ to its binding site. Mutagenesis and structural data rule out an alternative path through the pore defined by the 180 and 220 loops. We suggest that the active site communicates allosterically with the Na+ site through a network of H-bonded water molecules that embeds the primary specificity pocket. Perturbation of the mobility of S195 and its H-bonding capabilities alters interaction with this network and influences the kinetics of Na+ binding and allosteric transduction. These findings have general mechanistic relevance for Na+-activated proteases and allosteric enzymes.

Unraveling binding mechanism and kinetics of macrocyclic Gαq protein inhibitors

G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gαq protein family. We recently discovered that radiolabeled FR and YM display strongly divergent residence times, which translates into significantly longer antiasthmatic effects of FR. The present study is aimed at investigating the molecular basis for this observed disparity. Based on docking studies, we mutated amino acid residues of the Gαq protein predicted to interact with FR or YM, and recombinantly expressed the mutated Gαq proteins in cells in which the native Gαq proteins had been knocked out by CRISPR-Cas9. Both radioligands showed similar association kinetics, and their binding followed a conformational selection mechanism, which was rationalized by molecular dynamics simulation studies. Several mutations of amino acid residues near the putative binding site of the “lipophilic anchors” of FR, especially those predicted to interact with the isopropyl group present in FR but not in YM, led to dramatically accelerated dissociation kinetics. Our data indicate that the long residence time of FR depends on lipophilic interactions within its binding site. The observed structure-kinetic relationships point to a complex binding mechanism of FR, which likely involves snap-lock- or dowel-like conformational changes of either ligand or protein, or both. These experimental data will be useful for the design of compounds with a desired residence time, a parameter that has now been recognized to be of utmost importance in drug development.

Conformational Features of Ras: Key Hydrogen-Bonding Interactions of Gln61 in the Intermediate State during GTP Hydrolysis.

The Ras protein is one of the most important drug targets for battling cancers. To effectively design novel drugs of Ras, we characterize here its conformational ensembles for the hydrolysis intermediate state RasGDP·Pi and the product state RasGDP by extensive replica-exchange molecular dynamics simulations. Several substates for RasGDP·Pi have been identified, while structural analyses have revealed an unrecognized hydrogen-bonding network that stabilizes the hydrolysis intermediate state. More interestingly, Gln61, which is involved in numerous oncogenic mutations, was found to be engaged in this hydrogen-bonding network, adopting a specific conformation that always points to Pi in contrast to that in the RasGTP state. The simulations also reveal that RasGDP has more than one substate, suggesting a conformational selection mechanism for the interaction between Ras and the guanine nucleotide exchange factors (GEFs). These findings offer new opportunities for the drug design of Ras by stabilizing the hydrolysis intermediate or disrupting its interaction with the GEFs.

Microsecond Folding of preQ1 Riboswitch and Its Biological Significance Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.

Riboswitches are regulatory elements of bacterial mRNA which function with conformational switching upon binding of specific cellular metabolites. In particular, transcriptional riboswitches regulate gene expression kinetically through the conformational change of the aptamer domain. In this study, we investigate the conformational dynamics and ligand binding mechanisms of the aptamer domain of a transcriptional prequeuosine (preQ1) riboswitch from Bacillus subtilis using two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) with microsecond time resolution. The obtained time-resolved single-molecule data indicate that the aptamer domain undergoes folding/unfolding including three forms, which are attributed to hairpin (O), pseudoknot-like (pF), and H-type pseudoknot (fF) structures. It is found that a cofactor, Mg2+, binds only to the fF form with the conformational selection mechanism. In contrast, it is indicated that the ligand, preQ1, binds to the O form with the induced-fit mechanism and significantly accelerates the microsecond O → pF folding process. It is also shown that the binding with preQ1 substantially stabilizes the fF form that is generated from the pF form with a long time constant (>10 ms). Combining these results with the results of a former smFRET study on the slower time scale, we obtain an overall picture of the folding/unfolding dynamics of the aptamer domain as well as its energy landscape. On the basis of the picture obtained, we discuss the significance of the microsecond folding/unfolding of the aptamer domain for biological function of the riboswitch and propose the molecular mechanism of the gene expression controlled by the structural dynamics of the aptamer domain.

Mechanism of Ligand Recognition by Human ACE2 Receptor.

Angiotensin converting enzyme 2 (ACE2) plays a key role in renin–angiotensin system regulation and amino acid homeostasis. Human ACE2 acts as the receptor for severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. ACE2 is also widely expressed in epithelial cells of the lungs, heart, kidney, and pancreas. It is considered an important drug target for treating SARS-CoV-2 as well as pulmonary diseases, heart failure, hypertension, renal diseases, and diabetes. Despite the critical importance, the mechanism of ligand binding to the human ACE2 receptor remains unknown. Here, we have addressed this challenge through all-atom simulations using a novel ligand Gaussian accelerated molecular dynamics (LiGaMD) method. Microsecond time scale LiGaMD simulations have unprecedentedly captured multiple times of spontaneous binding and unbinding of a potent inhibitor MLN-4760 in the ACE2 receptor. With ligand far away in the unbound state, the ACE2 receptor samples distinct Open, Partially Open, Closed, and Fully Closed conformations. Upon ligand binding to the active site, conformational ensemble of the ACE2 receptor is biased toward the Closed state as observed in the X-ray experimental structure. The LiGaMD simulations thus suggest a conformational selection mechanism for ligand recognition by the highly flexible ACE2 receptor, which is expected to facilitate rational drug design targeting human ACE2 against coronaviruses and other related human diseases.

Solution-State Preorganization of Cyclic β-Hairpin Ligands Determines Binding Mechanism and Affinities for MDM2.

Understanding mechanisms of protein folding and binding is crucial to designing their molecular function. Molecular dynamics (MD) simulations and Markov state model (MSM) approaches provide a powerful way to understand complex conformational change that occurs over long time scales. Such dynamics are important for the design of therapeutic peptidomimetic ligands, whose affinity and binding mechanism are dictated by a combination of folding and binding. To examine the role of preorganization in peptide binding to protein targets, we performed massively parallel explicit-solvent MD simulations of cyclic β-hairpin ligands designed to mimic the p53 transactivation domain and competitively bind mouse double minute 2 homologue (MDM2). Disrupting the MDM2-p53 interaction is a therapeutic strategy to prevent degradation of the p53 tumor suppressor in cancer cells. MSM analysis of over 3 ms of aggregate trajectory data enabled us to build a detailed mechanistic model of coupled folding and binding of four cyclic peptides which we compare to experimental binding affinities and rates. The results show a striking relationship between the relative preorganization of each ligand in solution and its affinity for MDM2. Specifically, changes in peptide conformational populations predicted by the MSMs suggest that entropy loss upon binding is the main factor influencing affinity. The MSMs also enable detailed examination of non-native interactions which lead to misfolded states and comparison of structural ensembles with experimental NMR measurements. In contrast to an MSM study of p53 transactivation domain (TAD) binding to MDM2, MSMs of cyclic β-hairpin binding show a conformational selection mechanism. Finally, we make progress toward predicting accurate off rates of cyclic peptides using multiensemble Markov models (MEMMs) constructed from unbiased and biased simulated trajectories.

Role of bacterial RNA polymerase gate opening dynamics in DNA loading and antibiotics inhibition elucidated by quasi-Markov State Model

To initiate transcription, the holoenzyme (RNA polymerase [RNAP] in complex with σ factor) loads the promoter DNA via the flexible loading gate created by the clamp and β-lobe, yet their roles in DNA loading have not been characterized. We used a quasi-Markov State Model (qMSM) built from extensive molecular dynamics simulations to elucidate the dynamics of Thermus aquaticus holoenzyme's gate opening. We showed that during gate opening, β-lobe oscillates four orders of magnitude faster than the clamp, whose opening depends on the Switch 2's structure. Myxopyronin, an antibiotic that binds to Switch 2, was shown to undergo a conformational selection mechanism to inhibit clamp opening. Importantly, we reveal a critical but undiscovered role of β-lobe, whose opening is sufficient for DNA loading even when the clamp is partially closed. These findings open the opportunity for the development of antibiotics targeting β-lobe of RNAP. Finally, we have shown that our qMSMs, which encode non-Markovian dynamics based on the generalized master equation formalism, hold great potential to be widely applied to study biomolecular dynamics.

Structural mechanism underlying ligand binding and activation of PPARγ

Ligands bind to an occluded orthosteric ligand-binding pocket within the nuclear receptor ligand-binding domain. Molecular simulations have revealed theoretical ligand entry/exit pathways to the orthosteric pocket; however, it remains unclear whether ligand binding proceeds through induced fit or conformational selection mechanisms. Here, using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and surface plasmon resonance analysis, we provide evidence that structurally distinct agonists bind peroxisome proliferator-activated receptor γ (PPARγ) via a two-step induced fit mechanism involving an initial fast kinetic step followed by a slow conformational change. The agonist encounter complex binding pose is suggested in crystal structures where ligands bind to a surface pore suggested as a ligand entry site in molecular simulations. Our findings suggest an activation mechanism for PPARγ whereby agonist binding occurs through an initial encounter complex followed by a transition of the ligand into the final binding pose within the orthosteric pocket, inducing a transcriptionally active conformation.

Lipid regulation of hERG1 channel function

The lipid regulation of mammalian ion channel function has emerged as a fundamental mechanism in the control of electrical signalling and transport specificity in various cell types. In this work, we combine molecular dynamics simulations, mutagenesis, and electrophysiology to provide mechanistic insights into how lipophilic molecules (ceramide-sphingolipid probe) alter gating kinetics and K+ currents of hERG1. We show that the sphingolipid probe induced a significant left shift of activation voltage, faster deactivation rates, and current blockade comparable to traditional hERG1 blockers. Microseconds-long MD simulations followed by experimental mutagenesis elucidated ceramide specific binding locations at the interface between the pore and voltage sensing domains. This region constitutes a unique crevice present in mammalian channels with a non-swapped topology. The combined experimental and simulation data provide evidence for ceramide-induced allosteric modulation of the channel by a conformational selection mechanism.

Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection.

Short, structured fragments of non-coding mRNA may act as molecular switches upon binding specific ligands, regulating the translation of proteins encoded downstream this mRNA sequence. One switch, called riboswitch N1, is regulated by aminoglycosides such as neomycin. Nucleobase mutations in the apical loop, although distant from the binding pocket, significantly affect neomycin affinity and riboswitch regulatory efficiency. To explain this influence, we conducted molecular dynamics simulations using generalized replica exchange with solute tempering (gREST). Translation assay of a reporter protein in a yeast system shows that mutating A17 to G in the riboswitch apical loop reduces 6-fold the translation regulation efficiency of the mutant. Indeed, simulations of the unbound riboswitch show that G17 frequently stacks with base 7, while base 8 is stabilized towards the binding site in a way that it may interfere with the conformational selection mechanism and decrease riboswitch regulatory activity. In the riboswitch complexes, this single-point A to G mutation disrupts a strong hydrogen bond between nucleotides 5 and 17 and, instead, a new hydrogen bond between residue 17 and neomycin is created. This change forces neomycin to occupy a slightly shifted position in the binding pocket, which increases neomycin flexibility. Our simulations of the U14C mutation suggest that the riboswitch complex with neomycin is more stable if cytosine 14 is protonated. A hydrogen bond between the RNA phosphate and protonated cytosine appears as the stabilizing factor. Also, based on the cell-free translation assay and isothermal titration calorimetry experiments, mutations of nucleotides 14 and 15 affect only slightly the riboswitch ability to bind the ligand and its activity. Indeed, the simulation of the unbound U15A mutant suggests conformations preformed for ligand binding, which may explain slightly higher regulatory activity of this mutant. Overall, our results corroborate the in vivo and in vitro experiments on the N1 riboswitch-neomycin system, detail the relationship between nucleobase mutations and RNA dynamics, and reveal the conformations playing the major role in the conformational selection mechanism.

Conformational Selection Mechanism Provides Structural Insights into the Optimization of APC-Asef Inhibitors.

Metastasis is the major cause of death in colorectal cancer and it has been proven that inhibiting an interaction between adenomatous polyposis coli (APC) and Rho guanine nucleotide exchange factor 4 (Asef) efficaciously restrain metastasis. However, current inhibitors cannot achieve a satisfying effect in vivo and need to be optimized. In the present study, we applied molecular dynamics (MD) simulations and extensive analyses to apo and holo APC systems in order to reveal the inhibitor mechanism in detail and provide insights into optimization. MD simulations suggested that apo APC takes on a broad array of conformations and inhibitors stabilize conformation selectively. Representative structures in trajectories show specific APC-ligand interactions, explaining the different binding process. The stability and dynamic properties of systems elucidate the inherent factors of the conformation selection mechanism. Binding free energy analysis quantitatively confirms key interface residues and guide optimization. This study elucidates the conformation selection mechanism in APC-Asef inhibition and provides insights into peptide-based drug design.

Conformational rearrangement during activation of a metabotropic glutamate receptor.

G protein-coupled receptors (GPCRs) relay information across cell membranes through conformational coupling between the ligand-binding domain and cytoplasmic signaling domain. In dimeric class C GPCRs, the mechanism of this process, which involves propagation of local ligand-induced conformational changes over 12 nm through three distinct structural domains, is unknown. Here, we used single-molecule FRET (smFRET) and live-cell imaging and found that metabotropic glutamate receptor 2 (mGluR2) interconverts between four conformational states, two of which were previously unknown, and activation proceeds through the conformational selection mechanism. Furthermore, the conformation of the ligand-binding domains and downstream domains are weakly coupled. We show that the intermediate states act as conformational checkpoints for activation and control allosteric modulation of signaling. Our results demonstrate a mechanism for activation of mGluRs where ligand binding controls the proximity of signaling domains, analogous to some receptor kinases. This design principle may be generalizable to other biological allosteric sensors.