Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) is associated with human disorders, including Arts syndrome, retinal dystrophy, and gouty arthritis. Recent studies have demonstrated that PRPS can form filamentous cytoophidia in eukaryotes. Here, we show that PRPS forms cytoophidia in prokaryotes both in vitro and in vivo. Moreover, we solve two distinct filament structures of E. coli PRPS at near-atomic resolution using Cryo-EM. The formation of the two types of filaments is controlled by the binding of different ligands. One filament type is resistant to allosteric inhibition. The structural comparison reveals conformational changes of a regulatory flexible loop, which may regulate the binding of the allosteric inhibitor and the substrate ATP. A noncanonical allosteric AMP/ADP binding site is identified to stabilize the conformation of the regulatory flexible loop. Our findings not only explore a new mechanism of PRPS regulation with structural basis, but also propose an additional layer of cell metabolism through PRPS filamentation.

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.

Glutamate dehydrogenase (GDH) is a key enzyme interlinking carbon and nitrogen metabolism. Recent discoveries of the GDH specific role in breast cancer, hyperinsulinism/hyperammonemia (HI/HA) syndrome, and neurodegenerative diseases have reinvigorated interest on GDH regulation, which remains poorly understood despite extensive and long standing studies. Notwithstanding the growing evidence of the complexity of allosteric network behind GDH regulation, identifications of allosteric factors and associated mechanisms are paramount to deepen our understanding of the complex dynamics that regulate GDH enzymatic activity. Combining structural analyses of cryo-electron microscopy data with molecular dynamic simulations, here we show that the cofactor NADH is a key player in the GDH regulation process. Our structural analysis indicates that, binding to the regulatory sites in proximity of the antenna region, NADH acts as a positive allosteric modulator by enhancing both the affinity of the inhibitor GTP binding and inhibition of GDH catalytic activity. We further show that the binding of GTP to the NADH-bound GDH activates a triangular allosteric network, interlinking the inhibitor with regulatory and catalytic sites. This allostery produces a local conformational rearrangement that triggers an anticlockwise rotational motion of interlinked alpha-helices with specific tilted helical extension. This structural transition is a fundamental switch in the GDH enzymatic activity. It introduces a torsional stress, and the associated rotational shift in the Rossmann fold closes the catalytic cleft with consequent inhibition of the deamination process. In silico mutagenesis examinations further underpin the molecular basis of HI/HA dominant mutations and consequent over-activity of GDH through alteration of this allosteric communication network. These results shed new light on GDH regulation and may lay new foundation in the design of allosteric agents.

Ataxin-3, the protein responsible for spinocerebellar ataxia type-3, is a cysteine protease that specifically cleaves poly-ubiquitin chains and participates in the ubiquitin proteasome pathway. The enzymatic activity resides in the N-terminal Josephin domain. An unusual feature of ataxin-3 is its low enzymatic activity especially for mono-ubiquitinated substrates and short ubiquitin chains. However, specific ubiquitination at lysine 117 in the Josephin domain activates ataxin-3 through an unknown mechanism. Here, we investigate the effects of K117 ubiquitination on the structure and enzymatic activity of the protein. We show that covalently linked ubiquitin rests on the Josephin domain, forming a compact globular moiety and occupying a ubiquitin binding site previously thought to be essential for substrate recognition. In doing so, ubiquitination enhances enzymatic activity by locking the enzyme in an activated state. Our results indicate that ubiquitin functions both as a substrate and as an allosteric regulatory factor. We provide a novel example in which a conformational switch controls the activity of an enzyme that mediates deubiquitination.

Our ability to rationally optimize allosteric regulation is limited by incomplete knowledge of the mutations that tune allostery. Are these mutations few or abundant, structurally localized or distributed? To examine this, we conducted saturation mutagenesis of a synthetic allosteric switch in which Dihydrofolate reductase (DHFR) is regulated by a blue-light sensitive LOV2 domain. Using a high-throughput assay wherein DHFR catalytic activity is coupled to E. coli growth, we assessed the impact of 1548 viable DHFR single mutations on allostery. Despite most mutations being deleterious to activity, fewer than 5% of mutations had a statistically significant influence on allostery. Most allostery disrupting mutations were proximal to the LOV2 insertion site. In contrast, allostery enhancing mutations were structurally distributed and enriched on the protein surface. Combining several allostery enhancing mutations yielded near-additive improvements to dynamic range. Our results indicate a path toward optimizing allosteric function through variation at surface sites.

HSUR1 and HSUR2, two noncoding RNAs expressed by the oncogenic Herpesvirus saimiri, bind host microRNAs miR-142-3p, miR-16, and miR-27 with different purposes. While binding of miR-27 to HSUR1 triggers the degradation of the microRNA, miR-16 is tethered by HSUR2 to target host mRNAs to repress their expression. Here we show that the interaction with miR-142-3p is required for the activity of both HSURs. Coimmunoprecipitation experiments revealed that miR-142-3p allosterically regulates the binding of miR-27 and miR-16 to HSUR1 and HSUR2, respectively. The binding of two different miRNAs to each HSUR is not cooperative. HSURs can be engineered to be regulated by other miRNAs, indicating that the identity of the binding miRNA is not important for HSUR regulation. Our results uncover a mechanism for allosteric regulation of noncoding RNA function and a previously unappreciated way in which microRNAs can regulate gene expression.

Recent work indicates a general architecture for proteins in which sparse networks of physically contiguous and coevolving amino acids underlie basic aspects of structure and function. These networks, termed sectors, are spatially organized such that active sites are linked to many surface sites distributed throughout the structure. Using the metabolic enzyme dihydrofolate reductase as a model system, we show that: (1) the sector is strongly correlated to a network of residues undergoing millisecond conformational fluctuations associated with enzyme catalysis, and (2) sector-connected surface sites are statistically preferred locations for the emergence of allosteric control in vivo. Thus, sectors represent an evolutionarily conserved "wiring" mechanism that can enable perturbations at specific surface positions to rapidly initiate conformational control over protein function. These findings suggest that sectors enable the evolution of intermolecular communication and regulation.

Histone lysine methyltransferases (HKMTs) are key regulators of many cellular processes. By definition, HKMTs catalyse the methylation of lysine residues in histone proteins. The enzymatic activities of HKMTs are under precise control, with their allosteric regulation emerging as a prevalent paradigm. We review the molecular mechanisms of allosteric regulation of HKMTs using well-studied histone H3 (K4, K9, K27 and K36) methyltransferases as examples. We discuss the current advances and future potential in targeting allosteric sites of HKMTs for drug development.

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.

Plastins/fimbrins are conserved actin-bundling proteins contributing to motility, cytokinesis and other cellular processes by organizing strikingly different actin assemblies as in aligned bundles and branched networks. We propose that this ability of human plastins stems from an allosteric communication between their actin-binding domains (ABD1/2) engaged in a tight spatial association. Here we show that ABD2 can bind actin three orders of magnitude stronger than ABD1, unless the domains are involved in an equally strong inhibitory engagement. A mutation mimicking physiologically relevant phosphorylation at the ABD1-ABD2 interface greatly weakened their association, dramatically potentiating actin cross-linking. Cryo-EM reconstruction revealed the ABD1-actin interface and enabled modeling of the plastin bridge and domain separation in parallel bundles. We predict that a strong and tunable allosteric inhibition between the domains allows plastins to modulate the cross-linking strength, contributing to remodeling of actin assemblies of different morphologies defining the unique place of plastins in actin organization.

ATM/Tel1 is an apical kinase that orchestrates the multifaceted DNA damage response. Mutations of ATM/Tel1 are associated with ataxia telangiectasia syndrome. Here, we report cryo-EM structures of symmetric dimer (4.1 Å) and asymmetric dimer (4.3 Å) of Saccharomyces cerevisiae Tel1. In the symmetric state, the side chains in Tel1 C-terminus (residues 1129-2787) are discernible and an atomic model is built. The substrate binding groove is completely embedded in the symmetric dimer by the intramolecular PRD and intermolecular LID domains. Point mutations in these domains sensitize the S. cerevisiae cells to DNA damage agents and hinder Tel1 activation due to reduced binding affinity for its activator Xrs2/Nbs1. In the asymmetric state, one monomer becomes more compact in two ways: the kinase N-lobe moves down and the Spiral of α-solenoid moves upwards, which resemble the conformational changes observed in active mTOR. The accessibility of the activation loop correlates with the synergistic conformational disorders in the TRD1-TRD2 linker, FATC and PRD domains, where critical post-translational modifications and activating mutations are coincidently condensed. This study reveals a tunable allosteric network in ATM/Tel1, which is important for substrate recognition, recruitment and efficient phosphorylation.

The development of new synthetic biology circuits for biotechnology and medicine requires deeper mechanistic insight into allosteric transcription factors (aTFs). Here we studied the aTF UxuR, a homodimer of two domains connected by a highly flexible linker region. To explore how ligand binding to UxuR affects protein dynamics we performed molecular dynamics simulations in the free protein, the aTF bound to the inducer D-fructuronate or the structural isomer D-glucuronate. We then validated our results by constructing a sensor plasmid for D-fructuronate in Escherichia coli and performed site-directed mutagenesis. Our results show that zinc coordination is necessary for UxuR function since mutation to alanines prevents expression de-repression by D-fructuronate. Analyzing the different complexes, we found that the disordered linker regions allow the N-terminal domains to display fast and large movements. When the inducer is bound, UxuR can sample an open conformation with a more pronounced negative charge at the surface of the N-terminal DNA binding domains. In opposition, in the free and D-glucuronate bond forms the protein samples closed conformations, with a more positive character at the surface of the DNA binding regions. These molecular insights provide a new basis to harness these systems for biological systems engineering.

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. The reaction occurs in two separate catalytic domains, coupled by the long-range translocation of a biotinylated carrier domain (BCCP). Here, we use a series of hybrid PC enzymes to examine multiple BCCP translocation pathways in PC. These studies reveal that the BCCP domain of PC adopts a wide range of translocation pathways during catalysis. Furthermore, the allosteric activator, acetyl CoA, promotes one specific intermolecular carrier domain translocation pathway. These results provide a basis for the ordered thermodynamic state and the enhanced carboxyl group transfer efficiency in the presence of acetyl CoA, and reveal that the allosteric effector regulates enzyme activity by altering carrier domain movement. Given the similarities with enzymes involved in the modular synthesis of natural products, the allosteric regulation of carrier domain movements in PC is likely to be broadly applicable to multiple important enzyme systems.

Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3 (DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tail modifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation.

Control of enzyme allosteric regulation is required to drive metabolic flux toward desired levels. Although the three-dimensional (3D) structures of many enzyme-ligand complexes are available, it is still difficult to rationally engineer an allosterically regulatable enzyme without decreasing its catalytic activity. Here, we describe an effective strategy to deregulate the allosteric inhibition of enzymes based on the molecular evolution and physicochemical characteristics of allosteric ligand-binding sites. We found that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We applied our findings to design mutations in selected target residues that deregulate the allosteric activity of fructose-1,6-bisphosphatase (FBPase). Specifically, charged amino acids at less conserved positions were substituted with hydrophobic or neutral amino acids with similar sizes. The engineered proteins successfully diminished the allosteric inhibition of E. coli FBPase without affecting its catalytic efficiency. We expect that our method will aid the rational design of enzyme allosteric regulation strategies and facilitate the control of metabolic flux.

Human NAD-dependent isocitrate dehydrogenase or HsIDH3 catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the TCA cycle. HsIDH3 exists and functions as a heterooctamer composed of the αβ and αγ heterodimers, and is regulated allosterically and/or competitively by numerous metabolites including CIT, ADP, ATP, and NADH. In this work, we report the crystal structure of HsIDH3 containing a β mutant in apo form. In the HsIDH3 structure, the αβ and αγ heterodimers form the α2βγ heterotetramer via their clasp domains, and two α2βγ heterotetramers form the (α2βγ)2 heterooctamer through insertion of the N-terminus of the γ subunit of one heterotetramer into the back cleft of the β subunit of the other heterotetramer. The functional roles of the key residues at the allosteric site, the pseudo allosteric site, the heterodimer and heterodimer-heterodimer interfaces, and the N-terminal of the γ subunit are validated by mutagenesis and kinetic studies. Our structural and biochemical data together demonstrate that the allosteric site plays an important role but the pseudo allosteric site plays no role in the allosteric activation of the enzyme; the activation signal from the allosteric site is transmitted to the active sites of both αβ and αγ heterodimers via the clasp domains; and the N-terminal of the γ subunit plays a critical role in the formation of the heterooctamer to ensure the optimal activity of the enzyme. These findings reveal the molecular mechanism of the assembly and allosteric regulation of HsIDH3.

Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.

There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human β2-adrenergic receptor (β2AR) - a prototypical G protein-coupled receptor - is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates β2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5-7 of the receptor. The alternative mechanism, where the β2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of β2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions.

Glycogen synthase kinase 3β (GSK-3β) is a serine-threonine kinase belonging to the CMGC family that plays a key role in many biological processes, such as glucose metabolism, cell cycle regulation, and proliferation. Like most protein kinases, GSK-3β is regulated via multiple pathways and sites. We performed all-atom molecular dynamics simulations on the unphosphorylated and phosphorylated unbound GSK-3β and the phosphorylated GSK-3β bound to a peptide substrate, its product, and a derived inhibitor. We found that GSK-3β autophosphorylation at residue Tyr(216) results in widening of the catalytic groove, thereby facilitating substrate access. In addition, we studied the interactions of the phosphorylated GSK-3β with a substrate and peptide inhibitor located at the active site and observed higher affinity of the inhibitor to the kinase. Furthermore, we detected a potential remote binding site which was previously identified in other kinases. In agreement with experiments we observed that binding of specific peptides at this remote site leads to stabilization of the activation loop located in the active site. We speculate that this stabilization could enhance the catalytic activity of the kinase. We point to this remote site as being structurally conserved and suggest that the allosteric phenomenon observed here may occur in the protein kinase superfamily.

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5.