Subunit Interface Research Articles

The structural biology and dynamics of malate dehydrogenases.

Malate dehydrogenase (MDH) enzymes catalyze the reversible oxidoreduction of malate to oxaloacetate using NAD(P) as a cofactor. This reaction is vital for metabolism and the exchange of reducing equivalents between cellular compartments. There are more than 100 structures of MDH in the Protein Data Bank, representing species from archaea, bacteria, and eukaryotes. This conserved family of enzymes shares a common nucleotide-binding domain, substrate-binding domain, and subunits associate to form a dimeric or a tetrameric enzyme. Despite the variety of crystallization conditions and ligands in the experimental structures, the conformation and configuration of MDH are similar. The quaternary structure and active site dynamics account for most conformational differences in the experimental MDH structures. Oligomerization appears essential for activity despite each subunit having a structurally independent active site. There are two dynamic regions within the active site that influence substrate binding and possibly catalysis, with one of these regions adjoining the subunit interface. In this review, we introduce the reader to the general structural framework of MDH highlighting the conservation of certain features and pointing out unique differences that regulate MDH enzyme activity.

Two serial filters control P2X7 cation selectivity, Ser342 in the central pore and lateral acidic residues at the cytoplasmic interface.

The human P2X7 receptor (hP2X7R) is a homotrimeric cell surface receptor gated by extracellular ATP4- with two transmembrane helices per subunit, TM1 and TM2. A ring of three S342 residues, one from each pore-forming TM2 helix, located halfway across the membrane bilayer, functions to close and open the gate in the apo and ATP4--bound open states, respectively. The hP2X7R is selective for small inorganic cations, but can also conduct larger organic cations such as Tris+. Here, we show by voltage-clamp electrophysiology in Xenopus laevis oocytes that mutation of S342 residues to positively charged lysines decreases the selectivity for Na+ over Tris+, but maintains cation selectivity. Deep in the membrane, laterally below the S342 ring are nine acidic residues arranged as an isosceles triangle consisting of residues E14, D352, and D356 on each side, which do not move significantly during gating. When the E14K mutation is combined with lysine substitutions of D352 and/or D356, cation selectivity is lost and permeation of the small anion Cl- is allowed. Lysine substitutions of S342 together with D352 or E14 plus D356 in the acidic triangle convert the hP2X7R mutant to a fully Cl--selective ATP4--gated receptor. We conclude that the ion selectivity of wild-type hP2X7R is determined by two sequential filters in one single pathway: (i) a primary size filter, S342, in the membrane center and (ii) three cation filters lateral to the channel axis, one per subunit interface, consisting of a total of nine acidic residues at the cytoplasmic interface.

Thermostability and activity improvement in l-threonine aldolase through targeted mutations in V-shaped subunit

l-threonine aldolase (LTA) catalyzes the synthesis of β-hydroxy-α-amino acids, which are important chiral intermediates widely used in the fields of pharmaceuticals and pesticides. However, the limited thermostability of LTA hinders its industrial application. Furthermore, the trade-off between thermostability and activity presents a challenge in the thermostability engineering of this enzyme. This study proposes a strategy to regulate the rigidity of LTA's V-shaped subunit by modifying its opening and hinge regions, distant from the active center, aiming to mitigate the trade-off. With LTA from Bacillus nealsonii as targeted enzyme, a total of 25 residues in these two regions were investigated by directed evolution. Finally, mutant G85A/M207L/A12C was obtained, showing significantly enhanced thermostability with a 20 °C increase in T5060 to 66 °C, and specific activity elevated by 34 % at the optimum temperature. Molecular dynamics simulations showed that the newly formed hydrophobicity and hydrogen bonds improved the thermostability and boosted proton transfer efficiency. This work enhances the thermostability of LTA while preventing the loss of activity. It opens new avenues for the thermostability engineering of other industrially relevant enzymes with active center located at the interface of subunits or domains.

Identification of potent and reversible piperidine carboxamides that are species-selective orally active proteasome inhibitors to treat malaria

Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf_proteasome β5 active-site (Pfβ5). A potent analog (SW584) showed efficacy in a mouse model of human malaria after oral dosing. SW584 had a low propensity to generate resistance (minimum inoculum for resistance [MIR] >109) and was synergistic with dihydroartemisinin. Pf_proteasome purification was facilitated by His8-tag introduction onto β7. Inhibition of Pfβ5 correlated with parasite killing, without inhibiting human proteasome isoforms or showing cytotoxicity. The Pf_proteasome_SW584 cryoelectron microscopy (cryo-EM) structure showed that SW584 bound non-covalently distal from the catalytic threonine, in an unexplored pocket at the β5/β6/β3 subunit interface that has species differences between Pf and human proteasomes. Identification of a reversible, species selective, orally active series with low resistance propensity provides a path for drugging this essential target.

Revealing molecular determinants governing mambalgin-3 pharmacology at acid-sensing ion channel 1 variants

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.

Structural basis of the Meinwald rearrangement catalysed by styrene oxide isomerase

Membrane-bound styrene oxide isomerase (SOI) catalyses the Meinwald rearrangement—a Lewis-acid-catalysed isomerization of an epoxide to a carbonyl compound—and has been used in single and cascade reactions. However, the structural information that explains its reaction mechanism has remained elusive. Here we determine cryo-electron microscopy (cryo-EM) structures of SOI bound to a single-domain antibody with and without the competitive inhibitor benzylamine, and elucidate the catalytic mechanism using electron paramagnetic resonance spectroscopy, functional assays, biophysical methods and docking experiments. We find ferric haem b bound at the subunit interface of the trimeric enzyme through H58, where Fe(III) acts as the Lewis acid by binding to the epoxide oxygen. Y103 and N64 and a hydrophobic pocket binding the oxygen of the epoxide and the aryl group, respectively, position substrates in a manner that explains the high regio-selectivity and stereo-specificity of SOI. Our findings can support extending the range of epoxide substrates and be used to potentially repurpose SOI for the catalysis of new-to-nature Fe-based chemical reactions.

The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation

The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.

Interaction of fluralaner with binary model membranes. Potential implications in the selectivity for invertebrates/vertebrates

GABAA receptors (GABAA-R) are the target of widely used gabaergic insecticides such as fluralaner, a second generation non-competitive antagonist. Fluralaner blocks GABAA-R, binding to a pocket in the transmembrane subunit interface. Because of the location of its binding pocket, it is expected that fluralaner accesses to the receptor by previously partitioning into the bilayer. Therefore, differences in lipid composition of insect and mammal cells become relevant to determine the impact of membrane composition on the nonspecific selectivity of gabaergic insecticides. Insect cells show minimal concentration of cholesterol and they modulate membrane fluidity by regulating PE:PC ratios. On the other hand, vertebrate cholesterol concentration can be as high as 50%, depending on the type of cell. We used experimental Langmuir monolayer and Molecular Dynamics Simulations (MDS) to characterize the effect and interactions of the insecticide fluralaner with model membranes with varying concentrations of POPC:POPE and POPC:CHOL. Monolayer experiments showed that this insecticide reduces film rigidity. MDS showed that fluralaner is located at the interface between the acyl chain and the polar region of the membrane, but this location shifts at high CHOL concentrations towards the phosphate region. This is accompanied by a shift in the orientation of the fluralaner backbone, affecting fluralaner solubility. The presence of CHOL favors fluralaner aggregation, while POPE has the opposite effect, allowing its solubilization and the presence of individual insecticide molecules homogeneously dispersed. Our work suggests that differences in membrane composition could unspecifically influence the selectivity of gabaergic insecticides, modulating fluralaner solubility and fluralaner's access to GABAA-R.

Initial Amino Acid:Codon Assignments and Strength of Codon:Anticodon Binding.

The ribosome brings 3'-aminoacyl-tRNA and 3'-peptidyl-tRNAs together to enable peptidyl transfer by binding them in two major ways. First, their anticodon loops are bound to mRNA, itself anchored at the ribosomal subunit interface, by contiguous anticodon:codon pairing augmented by interactions with the decoding center of the small ribosomal subunit. Second, their acceptor stems are bound by the peptidyl transferase center, which aligns the 3'-aminoacyl- and 3'-peptidyl-termini for optimal interaction of the nucleophilic amino group and electrophilic ester carbonyl group. Reasoning that intrinsic codon:anticodon binding might have been a major contributor to bringing tRNA 3'-termini into proximity at an early stage of ribosomal peptide synthesis, we wondered if primordial amino acids might have been assigned to those codons that bind the corresponding anticodon loops most tightly. By measuring the binding of anticodon stem loops to short oligonucleotides, we determined that family-box codon:anticodon pairings are typically tighter than split-box codon:anticodon pairings. Furthermore, we find that two family-box anticodon stem loops can tightly bind a pair of contiguous codons simultaneously, whereas two split-box anticodon stem loops cannot. The amino acids assigned to family boxes correspond to those accessible by what has been termed cyanosulfidic chemistry, supporting the contention that these limited amino acids might have been the first used in primordial coded peptide synthesis.

Structure and function of GluN1-3A NMDA receptor excitatory glycine receptor channel.

N-methyl-d-aspartate receptors (NMDARs) and other ionotropic glutamate receptors (iGluRs) mediate most of the excitatory signaling in the mammalian brains in response to the neurotransmitter glutamate. Uniquely, NMDARs composed of GluN1 and GluN3 are activated exclusively by glycine, the neurotransmitter conventionally mediating inhibitory signaling when it binds to pentameric glycine receptors. The GluN1-3 NMDARs are vital for regulating neuronal excitability, circuit function, and specific behaviors, yet our understanding of their functional mechanism at the molecular level has remained limited. Here, we present cryo-electron microscopy structures of GluN1-3A NMDARs bound to an antagonist, CNQX, and an agonist, glycine. The structures show a 1-3-1-3 subunit heterotetrameric arrangement and an unprecedented pattern of GluN3A subunit orientation shift between the glycine-bound and CNQX-bound structures. Site-directed disruption of the unique subunit interface in the glycine-bound structure mitigated desensitization. Our study provides a foundation for understanding the distinct structural dynamics of GluN3 that are linked to the unique function of GluN1-3 NMDARs.

Novel α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) potentiator LT-102: A promising therapeutic agent for treating cognitive impairment associated with schizophrenia.

We aimed to evaluate the potential of a novel selective α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) potentiator, LT-102, in treating cognitive impairments associated with schizophrenia (CIAS) and elucidating its mechanism of action. The activity of LT-102 was examined by Ca2+ influx assays and patch-clamp in rat primary hippocampal neurons. The structure of the complex was determined by X-ray crystallography. The selectivity of LT-102 was evaluated by hERG tail current recording and kinase-inhibition assays. The electrophysiological characterization of LT-102 was characterized by patch-clamp recording in mouse hippocampal slices. The expression and phosphorylation levels of proteins were examined by Western blotting. Cognitive function was assessed using the Morris water maze and novel object recognition tests. LT-102 is a novel and selective AMPAR potentiator with little agonistic effect, which binds to the allosteric site formed by the intradimer interface of AMPAR's GluA2 subunit. Treatment with LT-102 facilitated long-term potentiation in mouse hippocampal slices and reversed cognitive deficits in a phencyclidine-induced mouse model. Additionally, LT-102 treatment increased the protein level of brain-derived neurotrophic factor and the phosphorylation of GluA1 in primary neurons and hippocampal tissues. We conclude that LT-102 ameliorates cognitive impairments in a phencyclidine-induced model of schizophrenia by enhancing synaptic function, which could make it a potential therapeutic candidate for CIAS.

The Cyclic Imine Core Common to the Marine Macrocyclic Toxins Is Sufficient to Dictate Nicotinic Acetylcholine Receptor Antagonism.

Macrocyclic imine phycotoxins are an emerging class of chemical compounds associated with harmful algal blooms and shellfish toxicity. Earlier binding and electrophysiology experiments on nAChR subtypes and their soluble AChBP surrogates evidenced common trends for substantial antagonism, binding affinities, and receptor-subtype selectivity. Earlier, complementary crystal structures of AChBP complexes showed that common determinants within the binding nest at each subunit interface confer high-affinity toxin binding, while distinctive determinants from the flexible loop C, and either capping the nest or extending toward peripheral subsites, dictate broad versus narrow receptor subtype selectivity. From these data, small spiroimine enantiomers mimicking the functional core motif of phycotoxins were chemically synthesized and characterized. Voltage-clamp analyses involving three nAChR subtypes revealed preserved antagonism for both enantiomers, despite lower subtype specificity and binding affinities associated with faster reversibility compared with their macrocyclic relatives. Binding and structural analyses involving two AChBPs pointed to modest affinities and positional variability of the spiroimines, along with a range of AChBP loop-C conformations denoting a prevalence of antagonistic properties. These data highlight the major contribution of the spiroimine core to binding within the nAChR nest and confirm the need for an extended interaction network as established by the macrocyclic toxins to define high affinities and marked subtype specificity. This study identifies a minimal set of functional pharmacophores and binding determinants as templates for designing new antagonists targeting disease-associated nAChR subtypes.

Selective Potentiation of the (α4)3(β2)2 Nicotinic Acetylcholine Receptor Response by NS9283 Analogues.

NS9283, 3-(3-pyridyl)-5-(3-cyanophenyl)-1,2,4-oxadiazole, is a selective positive allosteric modulator of (α4)3(β2)2 nicotinic acetylcholine receptors (nAChRs). It has good subtype selective therapeutic potential afforded by its specific binding to the unique α4-α4 subunit interface present in the (α4)3(β2)2 nAChR. However, there is currently a lack of structure activity relationship (SAR) studies aimed at developing a class of congeners endowed with the same profile of activity that can help consolidate the druggability of the α4-α4 subunit interface. In this study, new NS9283 analogues were designed, synthesized, and characterized for their ability to selectively potentiate the ACh activity at heterologous (α4)3(β2)2 nAChRs vs nAChR subtypes (α4)2(β2)3, α5α4β2, and α7. With few exceptions, all the NS9283 analogues exerted positive modulation of the (α4)3(β2)2 nAChR ACh-evoked responses. Above all, those modified at the 3-cyanophenyl moiety by replacement with 3-nitrophenyl (4), 4-cyanophenyl (10), and N-formyl-4-piperidinyl (20) showed the same efficacy as NS9283, although with lower potency. Molecular dynamics simulations of NS9283 and some selected analogues highlighted consistency between potentiation activity and pose of the ligand inside the α4-α4 site with the main interaction being with the complementary (-) side and induction of a significant conformational change of the Trp156 residue in the principal (+) side.

The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center.

Translation termination is an essential cellular process, which is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins from ribosomes and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. However, the mechanism of action of SRI-41315 on eRF1 and translation is not known. Here we report cryo-EM structures showing that SRI-41315 acts as a metal-dependent molecular glue between the N domain of eRF1 responsible for stop codon recognition and the ribosomal subunit interface near the decoding center. Retention of eRF1 on ribosomes by SRI-41315 leads to ribosome collisions, eRF1 ubiquitylation and a higher frequency of translation termination at near-cognate stop codons. Our findings reveal a new mechanism of release factor inhibition and additional implications for pharmacologically targeting eRF1.

Unveiling the molecular basis of lobeline's allosteric regulation of NMDAR: insights from molecular modeling

Neurological and psychiatric disorders contribute significantly to the global disease burden, adversely affecting the quality of life for both patients and their families. Impaired glutamatergic signaling is considered to be a major cause for most of the neurological and psychiatric disorders. Glutamate receptors are over activated in excitotoxic conditions, leading to dysregulation of Ca2+ homeostasis, triggering the production of free radicals and oxidative stress, mitochondrial dysfunction and eventually cell death. Excitotoxicity primarily results from the overactivity of NMDARs, a subtype of ionotropic glutamate receptors, due to their pronounced Ca2+ permeability and conductance characteristics. NMDAR antagonists are suggested to have therapeutic use as they can prevent excitotoxicity. Our previous studies demonstrated lobeline, an alkaloid, exerts neuroprotective action in excitotoxic conditions by blocking NMDAR. However, the atomic level interactions of lobeline with NMDAR was not characterized yet. Structural comparison of lobeline with a known NMDAR antagonist ifenprodil, followed by molecular docking and dynamics simulations revealed that lobeline could bind to the ifenprodil binding site i.e., in the heterodimer interface of GluN1-GluN2B subunits and exert ifenprodil like activities. By in silico structure guided modifications on lobeline and subsequent free energy calculations, we propose putative NMDAR antagonists derived from lobeline.

The Allosteric Regulation of Β-Ureidopropionase Depends on Fine-Tuned Stability of Active-Site Loops and Subunit Interfaces.

The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer-dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer-dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer-dimer interfaces are highly conserved between human and fruit fly enzymes.

Targeting the Structural Maturation Pathway of HIV-1 Reverse Transcriptase.

Formation of active HIV-1 reverse transcriptase (RT) proceeds via a structural maturation process that involves subdomain rearrangements and formation of an asymmetric p66/p66' homodimer. These studies were undertaken to evaluate whether the information about this maturation process can be used to identify small molecule ligands that retard or interfere with the steps involved. We utilized the isolated polymerase domain, p51, rather than p66, since the initial subdomain rearrangements are largely limited to this domain. Target sites at subdomain interfaces were identified and computational analysis used to obtain an initial set of ligands for screening. Chromatographic evaluations of the p51 homodimer/monomer ratio support the feasibility of this approach. Ligands that bind near the interfaces and a ligand that binds directly to a region of the fingers subdomain involved in subunit interface formation were identified, and the interactions were further characterized by NMR spectroscopy and X-ray crystallography. Although these ligands were found to reduce dimer formation, further efforts will be required to obtain ligands with higher binding affinity. In contrast with previous ligand identification studies performed on the RT heterodimer, subunit interface surfaces are solvent-accessible in the p51 and p66 monomers, making these constructs preferable for identification of ligands that directly interfere with dimerization.

Inhibition of human glutathione transferase by catechin and gossypol: comparative structural analysis by kinetic properties, molecular docking and their efficacy on the viability of human MCF-7 cells.

Glutathione transferase Pi (GSTP1) expression is increased in many cancer types and is associated with multidrug resistance and apoptosis inhibition. Inhibitors of GSTP1-1 have the potential to overcome drug resistance and improve chemotherapy efficacy as adjuvant agents. This study investigated the effects of catechin and gossypol on human glutathione transferase Pi (GSTP1-1) activity and their cytotoxic effects on breast cancer cells (MCF-7) individually and in combination with tamoxifen (TAM). Gossypol effectively inhibited the enzyme with an IC50 value of 40μM, compared to 200μM for catechin. Gossypol showed stronger inhibition of GSTP1-1 activity (Ki = 63.3 ± 17.5μM) compared to catechin (Ki = 220 ± 44μM). Molecular docking analysis revealed their binding conformations to GSTP1-1, with gossypol binding at the subunit interface in an un-competitive manner and catechin showing mixed non-competitive inhibition. Gossypol had severe cytotoxic effects on both MCF-7 cells and normal BJ1 cells, while catechin had a weak cytotoxic effect on MCF-7 cells only. Combination therapy with TAM resulted in cytotoxicity of 27.3% and 35.2% when combined with catechin and gossypol, respectively. Gossypol showed higher toxicity to MCF-7 cells, but its strong effects on normal cells raised concerns about selectivity and potential side effects.

Cryo-EM structures reveal native GABAA receptor assemblies and pharmacology.

Type A γ-aminobutyric acid receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants1-3. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits4 and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem(ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone(APG)). Using cryo-electron microscopy (cryo-EM)analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.

A critical inter-subunit interaction for the transmission of the allosteric signal in the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase is a key regulatory enzyme involved in starch and glycogen synthesis in plants and bacteria, respectively. It has been hypothesized that inter-subunit communications are important for the allosteric effect in this enzyme. However, no specific interactions have been identified as part of the regulatory signal. The enzyme from Agrobacterium tumefaciens is a homotetramer allosterically regulated by fructose 6-phosphate and pyruvate. Three pairs of distinct subunit-subunit interfaces are present. Here we focus on an interface that features two symmetrical interactions between Arg11 and Asp141 from one subunit with residues Asp141 and Arg11 of the neighbor subunit, respectively. Previously, scanning mutagenesis showed that a mutation at the Arg11 position disrupted the activation of the enzyme. Considering the distance of these residues from the allosteric and catalytic sites, we hypothesized that the interaction between Arg11 and Asp141 is critical for allosteric signaling rather than effector binding. To prove our hypothesis, we mutated those two sites (D141A, D141E, D141N, D141R, R11D, and R11K) and performed kinetic and binding analysis. Mutations that altered the charge affected the regulation the most. To prove that the interaction per se (rather than the presence of specific residues) is critical, we partially rescued the R11D protein by introducing a second mutation (R11D/D141R). This could not restore the activator effect on kcat , but it did rescue the effect on substrate affinity. Our results indicate the critical functional role of Arg11 and Asp141 to relay the allosteric signal in this subunit interface.