Influence of zinc doping on the molecular biocompatibility of cadmium-based quantum dots: Insights from the interaction with trypsin.

Doping quantum dots (QDs) with extra element presents a promising future for their applications in the fields of environmental monitoring, commercial products and biomedical sciences. However, it remains unknown for the influence of doping on the molecular biocompatibility of QDs and the underlying mechanisms of the interaction between doped-QDs and protein molecules. Using the "one-pot" method, we synthesized N-acetyl-l-cysteine capped CdTe: Zn2+ QDs with higher fluorescence quantum yield, improved stability and better molecular biocompatibility compared with undoped CdTe QDs. Using digestive enzyme trypsin (TRY) as the protein model, the interactions of undoped QDs and Zn-doped QDs with TRY as well as the underlying mechanisms were investigated using multi-spectroscopy, isothermal titration calorimetry and dialysis techniques. Van der Waals forces and hydrogen bonds are the major driving forces in the interaction of both QDs with TRY, which leading to the loosening of protein skeleton and tertiary structural changes. Compared with undoped QDs, Zn-doped QDs bind less amount of TRY with a higher affinity and then release higher amount of Cd. Zn-doped QDs have a less stimulating impact on TRY activity by decreasing TRY binding and reducing Cd binding to TRY. Taken all together, Zn-doped QDs offer a safer alternative for the applications of QDs by reducing unwanted interactions with proteins and improving biocompatibility at the molecular level.

Destruction mechanisms of ozone over SARS-CoV-2.

In this pandemic SARS-CoV-2 crisis, any attempt to contain and eliminate the virus will also stop its spread and consequently decrease the risk of severe illness and death. While ozone treatment has been suggested as an effective disinfection process, no precise mechanism of action has been previously reported. This study aimed to further investigate the effect of ozone treatment on SARS-CoV-2. Therefore, virus collected from nasopharyngeal and oropharyngeal swab and sputum samples from symptomatic patients was exposed to ozone for different exposure times. The virus morphology and structure were monitored and analyzed through Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Atomic Absorption Spectroscopy (AAS), and ATR-FTIR. The obtained results showed that ozone treatment not only unsettles the virus morphology but also alters the virus proteins' structure and conformation through amino acid disturbance and Zn ion release from the virus non-structural proteins. These results could provide a clearer pathway for virus elimination and therapeutics preparation.

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Structural and kinetic insights into HIV-1 reverse transcriptase inhibition by farnesiferol C.

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is the key enzyme for the virus gene replication and the most important target for antiviral therapy. Toxicity, drug resistance and side effects have led to search for new antiviral agents. Farnesiferol C (FC) is a well-known biologically active sesquiterpene coumarin derivative from genus Ferula. The current study was designed to examine the impacts of FC on the structure and function of HIV-1 RT, using some theoretical and experimental methods. FC inhibited HIV-1RT activity via mixed inhibition mechanism (IC50 = 30 µM). Spectroscopic data showed some conformational changes in the secondary as well as tertiary structure of HIV-1RT following the interaction with FC. Results showed that FC could quench the intrinsic fluorescence emission of HIV-1RT through static quenching mechanism. Thermodynamic parameters revealed that hydrogen bondings and van der Waals forces are the major forces in the binding reaction and the low equilibrium constants (KD) value obtained from surface plasmon resonance data, confirmed the high affinity of FC for HIV-1RT. Molecular docking studies indicated that FC interacts with enzyme through hydrophobic pocket. Taken together, the outcomes of this research revealed that, sesquiterpene coumarines can be used to design natural remedies as anti-HIV agents.

Effects of metal ions on activity and structure of phenoloxidase in Penaeus vannamei.

Phenoloxidase (PO) is a typical metal enzyme, which requires metal ions as prosthetic groups to enable the full exertion of its activity. To study how metal ions affected the activity and structure of PO enzymes, while providing reference materials for in-depth investigations, we examined the effects of different metal ions (Cu2+, Zn2+, Mg2+, Ca2+, and Ba2+) on their activities. Furthermore, Cu2+ and Mg2+ were selected for further investigation through UV spectra, intrinsic fluorescence spectroscopy, AFM, and FTIR. It was revealed that Cu2+ had a more obvious effect on PO compared to Mg2+. The PO could be activated when the concentrations of Cu2+ and Mg2+ were lower than 10-3 and 10-2 mol/L, respectively, and maximum PO activities (182.14% and 141.02%) were observed at 10-4 mol/L concentrations of Cu2+ and Mg2+. When the concentrations of Cu2+ and Mg2+ were higher than 10-2 and 10-1 mol/L, the activities PO were inhibited. The results of the UV-vis and fluorescence spectra revealed that Cu2+ shaped the tertiary structure of PO, whereas the effect of Mg2+ was slight. The AFM results demonstrated that high concentrations of Cu2+ and Mg2+ resulted in PO aggregation. FTIR analysis indicated that the total content of PO α-helices and ß-sheets decreased with higher concentrations of Cu2+ and Mg2+.

Solid Lipid Microparticles for Oral Delivery of Catalase: Focus on the Protein Structural Integrity and Gastric Protection.

Protein inactivation either during the production process or along the gastrointestinal tract is the major problem associated with the development of oral delivery systems for biological drugs. This work presents an evaluation of the structural integrity and the biological activity of a model protein, catalase, after its encapsulation in glyceryl trimyristate-based solid lipid microparticles (SLMs) obtained by the spray congealing technology. Circular dichroism and fluorescence spectroscopies were used to assess the integrity of catalase released from SLMs. The results confirmed that no conformational change occurred during the production process and both the secondary and tertiary structures were retained. Catalase is highly sensitive to temperature and undergoes denaturation above 60 °C; nevertheless, spray congealing allowed the retention of most biological activity due to the loading of the drug at the solid state, markedly reducing the risk of denaturation. Catalase activity after exposure to simulated gastric conditions (considering both acidic pH and the presence of gastric digestive hydrolases) ranged from 35 to 95% depending on the carrier: increasing of both the fatty acid chain length and the degree of substitution of the glyceride enhanced residual enzyme activity. SLMs allowed the protein release in a simulated intestinal environment and were not cytotoxic against HT29 cells. In conclusion, the encapsulation of proteins into SLMs by spray congealing might be a promising strategy for the formulation of nontoxic and inexpensive oral biotherapeutic products.

Oligosaccharides act as the high efficiency stabilizer for ß-galactosidase under heat treatment.

ß-Galactosidase (ß-Gal) as dietary supplement has the ability to alleviate symptoms of lactose intolerance. This study investigated the ability of oligosaccharides to protect ß-Gal against heat stress. Four kinds of oligosaccharides including Isomalto-oligosaccharides (IMO), Xylo-oligosaccharides (XOS), Konjac-oligosaccharides (KOS), and Mycose significantly increased the activity retention of ß-Gal under heat treatment. The results of three assays including circular dichroism, fluorescence, and Fourier transform infrared spectroscopy (FTIR) illustrated that these oligosaccharides could stabilize the secondary and tertiary structure of ß-Gal under thermal conditions through hydrogen bond interaction. Unlike these four oligosaccharides, Chito-oligosaccharides (COS) changed the secondary and tertiary structure of ß-Gal, thus decreasing its activity retention rate. Under heat treatment, the activity retention rate of ß-Gal with optimal composition (30% IMO, w/v and 40% XOS, w/v) reached 82.1%, which was significantly higher than that of the native ß-Gal (the activity retention rate of 20%). This study provides an insight into the mechanism by which sugar stabilizes protein under heat stress and offers guidance for application of liquid lactase to food industry.

Inhibitory effects of a sulfated polysaccharide isolated from edible red alga Bangia fusco-purpurea on α-amylase and α-glucosidase.

In this study, a sulfated polysaccharide (BFP) was isolated from the edible red alga Bangia fusco-purpurea. Gel-filtration and thin layer chromatographically analyses suggested that BFP was a homogenous polysaccharide. The chemical structural analysis revealed that BFP mainly consisted of galactose together with a small amount of uronic acid, mannose, and glucose. Its molecular mass was estimated to be 133.18 kDa by high-performance liquid chromatography (HPLC) analysis. BFP inhibited α-amylase and α-glucosidase in a concentration-dependent manner. The IC50 values of BFP against α-amylase and α-glucosidase were estimated to be 1.26 ± 0.11 mg/mL and 1.34 ± 0.07 mg/mL, respectively. Kinetic analyses suggested that BFP showed competitive and non-competitive inhibition against α-amylase and α-glucosidase, respectively. Circular dichroism spectral and fluorescence spectral analyses suggested that BFP affects the conformational structures of these enzymes, which may lead to the inhibition of the enzymatic activities. Abbreviations: Ara: D-arabinose; AnGal: anhydro-L-galactose residues; CD spectroscopy: Circular Dichroism spectroscopy; DNS: dinitrosalicylic acid; FT-IR: fourier transform infrared spectra; Fuc: L-fucose; Gal: D-galactose; Glc: D-glucose; GlcA: D-Glucuronic acid; HPLC: high performance liquid chromatography; Man: D-mannose; pNPG: p-nitrophenyl-α-D-glucoside; TFA: trifluoroacetic acid; TLC: thin-layer chromatography; PMP: 1-phenyl-3-methyl-5-pyrazolone; Xyl: D-xylose.

Characterisation of denatured states of sensory rhodopsin II by solution-state NMR.

Sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis, is a novel model system for membrane protein folding studies. Recently, the SDS-denatured states and the kinetics for reversible unfolding of pSRII have been investigated, opening the door to the first detailed characterisation of denatured states of a membrane protein by solution-state nuclear magnetic resonance (NMR) using uniformly 15N-labelled pSRII. SDS denaturation and acid denaturation of pSRII both lead to fraying of helix ends but otherwise small structural changes in the transmembrane domain, consistent with little changes in secondary structure and disruption of the retinal-binding pocket and tertiary structure. Widespread changes in the backbone amide dynamics are detected in the form of line broadening, indicative of µs-to-ms timescale conformational exchange in the transmembrane region. Detailed analysis of chemical shift and intensity changes lead to high-resolution molecular insights on structural and dynamics changes in SDS- and acid-denatured pSRII, thus highlighting differences in the unfolding pathways under the two different denaturing conditions. These results will form the foundation for furthering our understanding on the folding and unfolding pathways of retinal-binding proteins and membrane proteins in general, and also for investigating the importance of ligand-binding in the folding pathways of other ligand-binding membrane proteins, such as GPCRs.

Folding-function relationship of the most common cystic fibrosis-causing CFTR conductance mutants.

Cystic fibrosis is caused by mutations in the CFTR gene, which are subdivided into six classes. Mutants of classes III and IV reach the cell surface but have limited function. Most class-III and class-IV mutants respond well to the recently approved potentiator VX-770, which opens the channel. We here revisited function and folding of some class-IV mutants and discovered that R347P is the only one that leads to major defects in folding. By this criterion and by its functional response to corrector drug VX-809, R347P qualifies also as a class-II mutation. Other class-IV mutants folded like wild-type CFTR and responded similarly to VX-809, demonstrating how function and folding are connected. Studies on both types of defects complement each other in understanding how compounds improve mutant CFTR function. This provides an attractive unbiased approach for characterizing mode of action of novel therapeutic compounds and helps address which drugs are efficacious for each cystic fibrosis disease variant.

Protein Environment: A Crucial Triggering Factor in Josephin Domain Aggregation: The Role of 2,2,2-Trifluoroethanol.

The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially α-helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain.

Correctors of the Major Cystic Fibrosis Mutant Interact through Membrane-Spanning Domains.

The most common cystic fibrosis causing mutation is deletion of phenylalanine at position 508 (F508del), a mutation that leads to protein misassembly with defective processing. Small molecule corrector compounds: VX-809 or Corr-4a (C4) partially restores processing of the major mutant. These two prototypical corrector compounds cause an additive effect on F508del/cystic fibrosis transmembrane conductance regulator (CFTR) processing, and hence were proposed to act through distinct mechanisms: VX-809 stabilizing the first membrane-spanning domain (MSD) 1, and C4 acting on the second half of the molecule [consisting of MSD2 and/or nucleotide binding domain (NBD) 2]. We confirmed the effect of VX-809 in enhancing the stability of MSD1 and showed that it also allosterically modulates MSD2 when coexpressed with MSD1. We showed for the first time that C4 stabilizes the second half of the CFTR protein through its action on MSD2. Given the allosteric effect of VX-809 on MSD2, we were prompted to test the hypothesis that the two correctors interact in the full-length mutant protein. We did see evidence supporting their interaction in the full-length F508del-CFTR protein bearing secondary mutations targeting domain:domain interfaces. Disruption of the MSD1:F508del-NBD1 interaction (R170G) prevented correction by both compounds, pointing to the importance of this interface in processing. On the other hand, stabilization of the MSD2:F508del-NBD1 interface (by introducing R1070W) led to a synergistic effect of the compound combination on the total abundance of both the immature and mature forms of the protein. Together, these findings suggest that the two correctors interact in stabilizing the complex of MSDs in F508del-CFTR.

The Differential Response to Ca2+ from Vertebrate and Invertebrate Calumenin Is Governed by a Single Amino Acid Residue.

Calumenin (Calu) is a well-conserved multi-EF-hand-containing Ca2+-binding protein. In this work, we focused on the alterations that calumenin has undergone during evolution. We demonstrate that vertebrate calumenin is significantly different from its invertebrate homologues with respect to its response to Ca2+ binding. Human calumenin (HsCalu1) is intrinsically unstructured in the Ca2+ free form and responds to Ca2+ with a dramatic gain in structure. Calumenin from Caenorhabditis elegans (CeCalu) is structured even in the apo form, with no conformational change upon binding of Ca2+. We decode this structural and functional distinction by identifying a single "Leu" residue-based switch located in the fourth EF-hand of HsCalu1, occupied by "Gly" in the invertebrate homologues. We demonstrate that replacing Leu with Gly (L150G) in HsCalu1 enables the protein to adopt a structural fold even in the Ca2+ free form, similar to CeCalu, leading to ligand compensation (adoption of structure in the absence of Ca2+). The fourth (of seven) EF-hand of HsCalu1 nucleates the structural fold of the protein depending on the switch residue (Gly or Leu). Our analyses reveal that the Leu that replaced Gly from fishes onward is absolutely conserved in higher vertebrates, while lower organisms have Gly, not only enlarging the scope of Ca2+-dependent structural transitions but also drawing a boundary between the invertebrate and vertebrate calumenin. The evolutionary selection of the switch residue strongly corroborates the change in the structure of the protein and its pleiotropic functions and seems like it can be extended to the presence or absence of a heart in that organism.

Mechanism of salt-induced activity enhancement of a marine-derived laccase, Lac15.

Laccase (benzenediol: oxygen oxidoreductases, EC1.10.3.2) is a multi-copper oxidase capable of oxidizing a variety of phenolic and other aromatic organic compounds. The catalytic power of laccase makes it an attractive candidate for potential applications in many areas of industry including biodegradation of organic pollutants and synthesis of novel drugs. Most laccases are vulnerable to high salt and have limited applications. However, some laccases are not only tolerant to but also activated by certain concentrations of salt and thus have great application potential. The mechanisms of salt-induced activity enhancement of laccases are unclear as yet. In this study, we used dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, intrinsic fluorescence emission, circular dichroism, ultraviolet-visible light absorption, and an enzymatic assay to investigate the potential correlation between the structure and activity of the marine-derived laccase, Lac15, whose activity is promoted by low concentrations of NaCl. The results showed that low concentrations of NaCl exert little influence on the protein structure, which was partially folded in the absence of the salt; moreover, the partially folded rather than the fully folded state seemed to be favorable for enzyme activity, and this partially folded state was distinctive from the so-called 'molten globule' occasionally observed in active enzymes. More data indicated that salt might promote laccase activity through mechanisms involving perturbation of specific local sites rather than a change in global structure. Potential binding sites for chloride ions and their roles in enzyme activity promotion are proposed.

Synthetic food additive dye "Tartrazine" triggers amorphous aggregation in cationic myoglobin.

Protein aggregation, a characteristic of several neurodegenerative diseases, displays vast conformational diversity from amorphous to amyloid-like aggregates. In this study, we have explored the interaction of tartrazine with myoglobin protein at two different pHs (7.4 and 2.0). We have utilized various spectroscopic techniques (turbidity, Rayleigh light scattering (RLS), intrinsic fluorescence, Congo Red and far-UV CD) along with microscopy techniques i.e. atomic force microscopy (AFM) and transmission electron microscopy (TEM) to characterize the tartrazine-induced aggregation in myoglobin. The results showed that higher concentrations of tartrazine (2.0-10.0mM) induced amorphous aggregation in myoglobin at pH 2.0 via electrostatic interactions. However, tartrazine was not able to induce aggregation in myoglobin at pH 7.4; because of strong electrostatic repulsion between myoglobin and tartrazine at this pH. The tartrazine-induced amorphous aggregation process is kinetically very fast, and aggregation occurred without the formation of a nucleus. These results proposed that the electrostatic interaction is responsible for tartrazine-induced amorphous aggregation. This study may help in the understanding of mechanistic insight of aggregation by tartrazine.

Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients.

Mutations in the C-terminus of human erythroid 5-aminolevulinate synthase (hALAS2), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are associated with two different blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA-causing mutations yield hALAS2 variants with decreased activity, while XLPP-causing mutations result in a gain-of-function of hALAS2. There are no specific treatments for XLPP. Isonicotinic acid hydrazide (isoniazid, INH), an antituberculosis agent, can cause sideroblastic anemia as a side-effect, by limiting PLP availability to hALAS2, via inhibition of pyridoxal kinase or reaction with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We hypothesized that INH also binds and directly inhibits hALAS2. Using fluorescence-activated cell sorting and confocal fluorescence microscopy, we demonstrate that INH reduces protoporphyrin IX levels in HeLa cells expressing either wild-type hALAS2 or XLPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) reversed the cellular inhibition of hALAS2 activity by INH. Steady-state kinetic analyses with purified hALAS2 indicated that INH directly inhibits the enzyme, noncompetitively or uncompetitively, with an apparent Ki of 1.2µM. Circular dichroism spectroscopy revealed that INH triggered tertiary structural changes in hALAS2 that altered the microenvironment of the PLP cofactor and hampered the association of PLP with apo-hALAS2. Treatment of four XLPP patients with INH (5mg·kg-1·day-1) over a six-month period was well tolerated but without statistically significant modification of PPIX levels. These results, taken together, permit us to further an INH inhibition kinetic mechanism for ALAS, which suggests the possible use of INH-derived drugs in treating patients with XLPP and potentially other protoporphyrin-accumulating porphyrias.

Characterization of folding intermediates during urea-induced denaturation of human carbonic anhydrase II.

Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a ∼29kDa, ß-sheet dominated monomeric protein of 259 amino acid residues. In the present study, the urea-induced denaturation of HCAII was carried out which was a tri-phasic process, i.e., N (native) ↔ XI ↔ XII ↔ D (denatured) with stable intermediates XI and XII populated around 2 and 4M urea, respectively. The far-UV CD was used to characterize the intermediate states (XI and XII) for secondary structural content, near-UV CD for tertiary structure, dynamic light scattering for hydrodynamic radius and ANS fluorescence spectroscopy for the presence of exposed hydrophobic patches. Based on these experiments, we concluded that urea-induced XI state has characteristics of molten globule state while XII state bears characteristics features of pre-molten globule state. Characterization of the intermediates on the folding pathway will contribute to a deeper understanding of the structure-function relationship of HCAII. Furthermore, this system may provide an excellent model to study urea stress and the strategies adopted by the organisms to combat such a stress.

Chimeric Glutamate Receptor Subunits Reveal the Transmembrane Domain Is Sufficient for NMDA Receptor Pore Properties but Some Positive Allosteric Modulators Require Additional Domains.

UNLABELLED: NMDA receptors are ligand-gated ion channels that underlie transmission at excitatory synapses and play an important role in regulating synaptic strength and stability. Functional NMDA receptors require two copies of the GluN1 subunit coassembled with GluN2 (and/or GluN3) subunits into a heteromeric tetramer. A diverse array of allosteric modulators can upregulate or downregulate NMDA receptor activity. These modulators include both synthetic compounds and endogenous modulators, such as cis-unsaturated fatty acids, 24(S)-hydroxycholesterol, and various neurosteroids. To evaluate the structural requirements for the formation and allosteric modulation of NMDA receptor pores, we have replaced portions of the rat GluN1, GluN2A, and GluN2B subunits with homologous segments from the rat GluK2 kainate receptor subunit. Our results with these chimeric constructs show that the NMDA receptor transmembrane domain is sufficient to account for most pore properties, but that regulation by some allosteric modulators requires additional cytoplasmic or extracellular domains. SIGNIFICANCE STATEMENT: Glutamate receptors mediate excitatory synaptic transmission by forming cation channels through the membrane that open upon glutamate binding. Although many compounds have been identified that regulate glutamate receptor activity, in most cases the detailed mechanisms that underlie modulation are poorly understood. To identify what parts of the receptor are essential for pore formation and sensitivity to allosteric modulators, we generated chimeric subunits that combined segments from NMDA and kainate receptors, subtypes with distinct pharmacological profiles. Surprisingly, our results identify separate domain requirements for allosteric potentiation of NMDA receptor pores by pregnenolone sulfate, 24(S)-hydroxycholesterol, and docosahexaenoic acid, three endogenous modulators derived from membrane constituents. Understanding where and how these compounds act on NMDA receptors should aid in designing better therapeutic agents.

A substrate-driven allosteric switch that enhances PDI catalytic activity.

Protein disulfide isomerase (PDI) is an oxidoreductase essential for folding proteins in the endoplasmic reticulum. The domain structure of PDI is a-b-b'-x-a', wherein the thioredoxin-like a and a' domains mediate disulfide bond shuffling and b and b' domains are substrate binding. The b' and a' domains are connected via the x-linker, a 19-amino-acid flexible peptide. Here we identify a class of compounds, termed bepristats, that target the substrate-binding pocket of b'. Bepristats reversibly block substrate binding and inhibit platelet aggregation and thrombus formation in vivo. Ligation of the substrate-binding pocket by bepristats paradoxically enhances catalytic activity of a and a' by displacing the x-linker, which acts as an allosteric switch to augment reductase activity in the catalytic domains. This substrate-driven allosteric switch is also activated by peptides and proteins and is present in other thiol isomerases. Our results demonstrate a mechanism whereby binding of a substrate to thiol isomerases enhances catalytic activity of remote domains.

Structural basis of Smoothened regulation by its extracellular domains.

Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.