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Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets 1-5 . More than 210 structures from more than 20 different TRP channels have been determined, and all are tetramers 4 . Despite this wealth of structures, many aspects concerning TRPV channels remain poorly understood, including the pore-dilation phenomenon, whereby prolonged activation leads to increased conductance, permeability to large ions and loss of rectification 6,7 . Here, we used high-speed atomic force microscopy (HS-AFM) to analyse membrane-embedded TRPV3 at the single-molecule level and discovered a pentameric state. HS-AFM dynamic imaging revealed transience and reversibility of the pentamer in dynamic equilibrium with the canonical tetramer through membrane diffusive protomer exchange. The pentamer population increased upon diphenylboronic anhydride (DPBA) addition, an agonist that has been shown to induce TRPV3 pore dilation. On the basis of these findings, we designed a protein production and data analysis pipeline that resulted in a cryogenic-electron microscopy structure of the TRPV3 pentamer, showing an enlarged pore compared to the tetramer. The slow kinetics to enter and exit the pentameric state, the increased pentamer formation upon DPBA addition and the enlarged pore indicate that the pentamer represents the structural correlate of pore dilation. We thus show membrane diffusive protomer exchange as an additional mechanism for structural changes and conformational variability. Overall, we provide structural evidence for a non-canonical pentameric TRP-channel assembly, laying the foundation for new directions in TRP channel research., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The main protease M pro , 3CL pro is an important target from coronaviruses. In spite of having 96% sequence identity among M pros from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 M pro so far, were found to have differential inhibitory effect on M pro of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of M pro from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 M pro in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo M pros from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in M pros . Moreover, we have also investigated the network connectivity on the quaternary structure of M pro and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development., (© 2021 Wiley Periodicals LLC.)

The selective targeting of protein-protein interactions remains a significant determinant for the proper modulation and regulation of cell apoptosis. Prototypic galectins such as human galectin-7 (GAL-7) are characterized by their ability to form homodimers that control the molecular fate of a cell by mediating subtle yet critical glycan-dependent interactions between pro- and anti-apoptotic molecular partners. Altering the structural architecture of GAL-7 can therefore result in resistance to apoptosis in various human cancer cells, further illustrating its importance in cell survival. In this study, we used a combination of biophysical and cellular assays to illustrate that binding of a water-soluble meso -tetraarylporphyrin molecule to GAL-7 induces protein oligomerization and modulation of GAL-7-induced apoptosis in human Jurkat T cells. Our results suggest that the integrity of the GAL-7 homodimer architecture is essential for its molecular function, in addition to providing an interesting porphyrin binding modulator for controlling apoptosis in mammalian cells.

Acid-sensing ion channel (ASIC) subunits 1a and 3 are highly expressed in central and peripheral sensory neurons, respectively. Endogenous biomolecule zinc plays a critical role in physiological and pathophysiological conditions. Here, we found that currents recorded from heterologously expressed ASIC1a/3 channels using the whole-cell patch-clamp technique were regulated by zinc with dual effects. Co-application of zinc dose-dependently potentiated both peak amplitude and the sustained component of heteromeric ASIC1a/3 currents; pretreatment with zinc between 3 to 100 µM exerted the same potentiation as co-application. However, pretreatment with zinc induced a significant inhibition of heteromeric ASIC1a/3 channels when zinc concentrations were over 250 µM. The potentiation of heteromeric ASIC1a/3 channels by zinc was pH dependent, as zinc shifted the pH dependence of ASIC1a/3 currents from a pH 50 of 6.54 to 6.77; whereas the inhibition of ASIC1a/3 currents by zinc was also pH dependent. Furthermore, we systematically mutated histidine residues in the extracellular domain of ASIC1a or ASIC3 and found that histidine residues 72 and 73 in both ASIC1a and ASIC3, and histidine residue 83 in the ASIC3 were responsible for bidirectional effects on heteromeric ASIC1a/3 channels by zinc. These findings suggest that histidine residues in the extracellular domain of heteromeric ASIC1a/3 channels are critical for zinc-mediated effects.

The antigen-antibody complex (AAC) has novel functions for immunomodulation, encouraging the application of diverse quaternary protein structures for vaccination. In this study, GA733 antigen and anti-GA733 antibody proteins were both co-expressed to obtain the AAC protein structures in a F1 plant obtained by crossing the plants expressing each protein. In F1 plant, the antigen and antibody assembled to form a large quaternary circular ACC structure (~30 nm). The large quaternary protein structures induced immune response to produce anticancer immunoglobulins G (IgGs) that are specific to the corresponding antigens in mouse. The serum containing the anticancer IgGs inhibited the human colorectal cancer cell growth in the xenograft nude mouse. Taken together, antigens and antibodies can be assembled to form AAC protein structures in plants. Plant crossing represents an alternative strategy for the formation of AAC vaccines that efficiently increases anticancer antibody production.

In this work, the effects of I 2 on the activities and conformational structures of digestive enzymes, trypsin and pepsin were studied. The results indicated that the enzyme activities were decreased to some extent in the presence of I 2 , especially trypsin. Upon gradual addition of I 2 , the intrinsic fluorescence quenching of trypsin and pepsin were observed by mainly static collision and hydrophobic forces. I 2 is more likely to cause the fluorescence quenching of trypsin than that of pepsin. Compared with pepsin, trypsin has a greater ability to bind with I 2 . The synchronous fluorescence spectral results indicated that I 2 induced the quaternary structure changes of trypsin/pepsin and changed the hydrophobicity of Tyr and Trp residues. In addition, molecular docking was used to obtain the binding mode and the various amino acid residues of trypsin and pepsin with I 2 . These investigations may constitute a solid work to further explain the process of migration and transformation of I 2 in digestive system., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript entitled “Evaluation of the binding mechanism of iodine with trypsin and pepsin:A spectroscopic and molecular docking”., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Protein kinase CK2, a heterotetrameric holoenzyme composed of two catalytic chains (CK2α) attached to a homodimer of regulatory subunits (CK2β), is a target for drug development for cancer therapy. Here, we describe the tetraiodobenzimidazole derivative ARC-3140, a bisubstrate inhibitor addressing the ATP site and the substrate-binding site of CK2 with extraordinary affinity (K i  = 84 pM). In a crystal structure of ARC-3140 in complex with CK2α, three copies of the inhibitor are visible, one of them at the CK2β interface of CK2α. Subsequent interaction studies based on microscale thermophoresis and fluorescence anisotropy changes revealed a significant impact of ARC-3140 and of its tetrabromo equivalent ARC-1502 on the CK2α/CK2β interaction. A structural inspection revealed that ARC-3140, unlike CK2β antagonists described so far, interferes with both sub-interfaces of the bipartite CK2α/CK2β interaction. Thus, ARC-3140 is a lead for the further development of highly effective compounds perturbating the quaternary structure of the CK2α 2 β 2 holoenzyme., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn's disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions.

Flaviviruses are emerging arthropod-borne viruses posing a great threat to human beings worldwide. The E dimer configuration of the flavivirus was prominent during viral assembly, maturation and entry. Neutralization antibodies targeting E dimer played the important role in controlling the flavivirus infection. Previously, the ideal drug target of small molecular inhibitors of JEV was viral proteases and polymerases. The crystal structure of JEV E protein showed a conserved pocket in it is important at membrane fusion step. Recently, a set of anti-virus drugs has been found by virtual screening. Here, we show that the fusion-loop pocket of JEV E protein was a conservative region and an ideal drug target. ChemDiv-3 from virtual screening as the lead compound was found to show a relatively modest inhibition effect for JEV in vitro and in vivo test and could interfere with the formation of JEV sE dimer. ChemDiv-3 interacts with the amino acid residues ASN 313, PRO 314, ALA 315, and VAL 323 in E protein via hydrogen bonds for occupation of the fusion-loop pocket. The key binding sites LYS 312, ALA 513 and THR 317 forming the fusion-loop pocket are the same and other auxiliary sites are similar among the flavivirus. Taken together, the fusion-loop pocket of the flavivirus could be one promising target for drug discovery., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.

The human Receptor for Advanced Glycation End Products (hRAGE) is a pattern recognition receptor implicated in inflammation and adhesion. It is involved in both innate and adaptive immunity. Its aberrant signaling is tied to the pathogenesis of diabetic complications, neurodegenerative disorders, and chronic inflammatory responses. Previous structural studies have focused on its extracellular domains with their canonical constant and variable Ig folds, and to a much lesser extent, the intrinsically disorder cytoplasmic domain. No experimental data are reported on the transmembrane domain, which is integral to signaling. We have constructed, expressed and purified the transmembrane domain attached to the cytoplasmic domain of hRAGE in E. coli. Multiple self-associated forms of these domains were observed in vitro. This pattern of mixed oligomers resembled previously reported in vivo forms of the complete receptor. The self-association of these two domains was further characterized using: SDS-PAGE, intrinsic tryptophan fluorescence and heteronuclear NMR spectroscopy. NMR conditions were assessed across time and temperature within micelles. Our data show that the transmembrane and cytoplasmic domains of hRAGE undergo dynamic oligomerizations that can occur in the absence of its extracellular domains or ligand binding. And, such associations are only partially disrupted even with prolonged incubation in strong detergents., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

Protein is a promising material for fabricating the biocompatible films used in the biomedical fields and food industry. Previously, we successfully prepared a water-insoluble albumin film possessing native albumin properties such as resistance to cell adhesion and drug-binding ability. Here, I quantitatively investigated the conformation of albumin in a film state using circular dichroism (CD) spectroscopy. The albumin film was prepared by crosslinking albumin with ethylene glycol diglycidyl ether (EGDE). CD measurements of albumin films revealed that approximately 70% of the α-helical structure was retained after film formation. Albumin molecules in the films acquired high stability. The conformation of albumin was completely retained even after heating at 100 °C for 1 h. For comparison, crosslinked albumin film was also prepared using glutaraldehyde (GA). Unlike EGDE-crosslinking, GA-crosslinking induced significant conformational changes in albumin; 46% of the α-helical structure was destroyed in GA-crosslinked albumin films. Cell adhesion studies showed that EGDE-crosslinked albumin film maintained the cell-nonadhesive property inherent in native albumin. This property was lost in GA-crosslinked albumin film, and cells adhesion occurred at a level comparable to that of cell culture dishes. These results indicate that EGDE-crosslinking is a useful method for preparing albumin films in which the native albumin structure and property are retained. The approach described here provides valuable information for creating protein films possessing high functionality., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The oligomeric organization of the voltage-dependent anion-selective channel (VDAC) and its interactions with hexokinase play integral roles in mitochondrially mediated apoptotic signaling. Various small to large assemblies of VDAC are observed in mitochondrial outer membranes, but they do not predominate in detergent-solubilized VDAC samples. In this study, a cholesterol analog, cholesteryl-hemisuccinate (CHS), was shown to induce the formation of detergent-soluble VDAC multimers. The various oligomeric states of VDAC induced by the addition of CHS were deciphered through an integrated biophysics approach using microscale thermophoresis, analytical ultracentrifugation, and size-exclusion chromatography small angle x-ray scattering. Furthermore, CHS stabilizes the interaction between VDAC and hexokinase (K d of 27 ± 6 μM), confirming the biological relevance of oligomers generated. Thus, sterols such as cholesterol in higher eukaryotes or ergosterol in fungi may regulate the VDAC oligomeric state and may provide a potential target for the modulation of apoptotic signaling by effecting VDAC-VDAC and VDAC-hexokinase interactions. In addition, the integrated biophysical approach described provides a powerful platform for the study of membrane protein complexes in solution., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Human mini-brains (MB) are cerebral organoids that recapitulate in part the complexity of the human brain in a unique three-dimensional in vitro model, yielding discrete brain regions reminiscent of the cerebral cortex. Specific proteins linked to neurodegenerative disorders are physiologically expressed in MBs, such as APP-derived amyloids (Aβ), whose physiological and pathological roles and interactions with other proteins are not well established in humans. Here, we demonstrate that neuroectodermal organoids can be used to study the Aβ accumulation implicated in Alzheimer's disease (AD). To enhance the process of protein secretion and accumulation, we adopted a chemical strategy of induction to modulate post-translational pathways of APP using an Amyloid-β Forty-Two Inducer named Aftin-5. Secreted, soluble Aβ fragment concentrations were analyzed in MB-conditioned media. An increase in the Aβ42 fragment secretion was observed as was an increased Aβ42/Aβ40 ratio after drug treatment, which is consistent with the pathological-like phenotypes described in vivo in transgenic animal models and in vitro in induced pluripotent stem cell-derived neural cultures obtained from AD patients. Notably in this context we observe time-dependent Aβ accumulation, which differs from protein accumulation occurring after treatment. We show that mini-brains obtained from a non-AD control cell line are responsive to chemical compound induction, producing a shift of physiological Aβ concentrations, suggesting that this model can be used to identify environmental agents that may initiate the cascade of events ultimately leading to sporadic AD. Increases in both Aβ oligomers and their target, the cellular prion protein (PrPC), support the possibility of using MBs to further understand the pathophysiological role that underlies their interaction in a human model. Finally, the potential application of MBs for modeling age-associated phenotypes and the study of neurological disorders is confirmed., Competing Interests: The authors have declared that no competing interests exist.

LysR-type transcriptional regulators (LTTRs) generally bind to target promoters in two conformations, depending on the availability of inducing ligands. OccR is an LTTR that regulates the octopine catabolism operon of Agrobacterium tumefaciens. OccR binds to a site located between the divergent occQ and occR promoters. Octopine triggers a conformational change that activates the occQ promoter, and does not affect autorepression. This change shortens the length of bound DNA and relaxes a high-angle DNA bend. Here, we describe the crystal structure of the ligand-binding domain (LBD) of OccR apoprotein and holoprotein. Pairs of LBDs form dimers with extensive hydrogen bonding, while pairs of dimers interact via a single helix, creating a tetramer interface. Octopine causes a 70° rotation of each dimer with respect to the opposite dimer, precisely at the tetramer interface. We modeled the DNA binding domain (DBD), linker helix and bound DNA onto the apoprotein and holoprotein. The two DBDs of the modeled apoprotein lie far apart and the bound DNA between them has a high-angle DNA bend. In contrast, the two DBDs of the holoprotein lie closer to each other, with a low DNA bend angle. This inter-dimer pivot fully explains earlier studies of this LTTR., (© 2018 John Wiley & Sons Ltd.)

The catecholaldehyde hypothesis posits that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediary metabolite of dopamine, is an autotoxin that challenges neuronal homeostasis in catecholaminergic neurons. DOPAL toxicity may involve protein modifications, such as oligomerization of α -synuclein (AS). Potential interactions between DOPAL and other proteins related to catecholaminergic neurodegeneration, however, have not been systemically explored. This study examined DOPAL-induced protein-quinone adduct formation ("quinonization") and protein oligomerization, ubiquitination, and aggregation in cultured MO3.13 human oligodendrocytes and PC12 rat pheochromocytoma cells and in test tube experiments. Using near-infrared fluorescence spectroscopy, we detected spontaneous DOPAL oxidation to DOPAL-quinone, DOPAL-induced quinonization of intracellular proteins in both cell lines, and DOPAL-induced quinonization of several proteins related to catecholaminergic neurodegeneration, including AS, the type 2 vesicular monoamine transporter, glucocerebrosidase, ubiquitin, and l-aromatic-amino-acid decarboxylase (LAAAD). DOPAL also oligomerized AS, ubiquitin, and LAAAD; inactivated LAAAD (IC 50 54 μ M); evoked substantial intracellular protein ubiquitination; and aggregated intracellular AS. Remarkably, N -acetylcysteine, which decreases DOPAL-quinone formation, attenuated or prevented all of these protein modifications and functional changes. The results fit with the proposal that treatments based on decreasing the formation and oxidation of DOPAL may slow or prevent catecholaminergic neurodegeneration., (U.S. Government work not protected by U.S. copyright.)

Acetyl-CoA carboxylase catalyses the ATP-dependent carboxylation of acetyl-CoA, a rate-limiting step in fatty acid biosynthesis 1,2 . Eukaryotic acetyl-CoA carboxylases are large, homodimeric multienzymes. Human acetyl-CoA carboxylase occurs in two isoforms: the metabolic, cytosolic ACC1, and ACC2, which is anchored to the outer mitochondrial membrane and controls fatty acid β-oxidation 1,3 . ACC1 is regulated by a complex interplay of phosphorylation, binding of allosteric regulators and protein-protein interactions, which is further linked to filament formation 1,4-8 . These filaments were discovered in vitro and in vivo 50 years ago 7,9,10 , but the structural basis of ACC1 polymerization and regulation remains unknown. Here, we identify distinct activated and inhibited ACC1 filament forms. We obtained cryo-electron microscopy structures of an activated filament that is allosterically induced by citrate (ACC-citrate), and an inactivated filament form that results from binding of the BRCT domains of the breast cancer type 1 susceptibility protein (BRCA1). While non-polymeric ACC1 is highly dynamic, filament formation locks ACC1 into different catalytically competent or incompetent conformational states. This unique mechanism of enzyme regulation via large-scale conformational changes observed in ACC1 has potential uses in engineering of switchable biosynthetic systems. Dissecting the regulation of acetyl-CoA carboxylase opens new paths towards counteracting upregulation of fatty acid biosynthesis in disease.

Alzheimer's disease (AD) is characterized by the presence of tau filaments in the brain whose structure was recently solved. The formation of AD filaments is routinely modeled in vitro by mixing tau with heparin. This study shows that heparin-induced tau filaments are markedly different from the AD filaments and are highly heterogeneous.

Fast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries 1 . This intrinsic heterogeneity in protein composition provides fine tunability in channel properties, which is essential to brain function, but frustrates structural and biophysical characterization. The α4β2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α- and β-subunits (2α:3β and 3α:2β). Both assemblies are functional and have distinct biophysical properties, and an imbalance in the ratio of assemblies is linked to both nicotine addiction 2,3 and congenital epilepsy 4,5 . Here we leverage cryo-electron microscopy to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to β2 were used to 'break' symmetry during particle alignment and to obtain high-resolution reconstructions of receptors of both stoichiometries in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor.

The present work deals with investigating the role of ionic interactions in the native conformation of BmGK by altering pH and salt concentration as well as by disruption of inter-subunit region. The study on structural and functional properties of BmGK as a function of pH showed that the secondary and tertiary elements of the protein were disturbed at low pH with loss of its native oligomerization and functional activity. High concentration of NaCl also changed the native conformation of BmGK with dissociation of its dimeric form. We also mutated dimeric interface of BmGK and identified intersubunit residues, Arg105 and Glu140, essential for dimer stability as double mutation at both positions hinders dimerization. The quaternary structure is found to be essential for full enzymatic activity and stability. In vitro results were supported by in silico molecular dynamics simulation studies through conformational stability analysis. Thus, the work carried out points toward new approach of targeting dimeric interface of BmGK in lieu of its similar active site region to its counterpart human enzyme. This may lead to the design of inhibitors targeted to key parasitic enzyme (BmGK) specifically., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å 2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor., Competing Interests: The authors declare no competing financial interests.

Calmodulin (CaM) is an essential protein present in all eukaryote cells, ranging from vertebrates to unicellular organisms. CaM is the most important Ca 2+ signalling protein, composed of two domains, N- and C-terminal domains, linked by a flexible central α-helix, and is responsible for the regulation of numerous calcium-mediated signalling pathways. Four calcium ions bind to CaM, changing its conformation and determining how it recognizes and regulates its cellular targets. The oxidation mechanism of native and denatured CaM, at a glassy carbon electrode, was investigated using differential pulse voltammetry and electrochemical impedance spectroscopy. Native and denatured CaM presented only one oxidation peak, related to the tyrosine amino acid residue oxidation. Calcium-induced calmodulin conformational change and the influence of Ca 2+ concentration on the electrochemical behaviour of CaM were evaluated, and significant differences, in the tyrosine amino acid residue peak potential and current, in the absence and in the presence of calcium ions, were observed. Gravimetric measurements were performed with a graphite coated piezoelectric quartz crystal with adsorbed CaM, and calcium aggregation by CaM was demonstrated., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Intracellular Ca 2+ signalling processes are fundamental to muscle contraction, neurotransmitter release, cell growth and apoptosis. Release of Ca 2+ from the intracellular stores is supported by a series of ion channels in sarcoplasmic or endoplasmic reticulum (SR/ER). Among them, two isoforms of the trimeric intracellular cation (TRIC) channel family, named TRIC-A and TRIC-B, modulate the release of Ca 2+ through the ryanodine receptor or inositol triphosphate receptor, and maintain the homeostasis of ions within SR/ER lumen. Genetic ablations or mutations of TRIC channels are associated with hypertension, heart disease, respiratory defects and brittle bone disease. Despite the pivotal function of TRIC channels in Ca 2+ signalling, their pore architectures and gating mechanisms remain unknown. Here we present the structures of TRIC-B1 and TRIC-B2 channels from Caenorhabditis elegans in complex with endogenous phosphatidylinositol-4,5-biphosphate (PtdIns(4,5)P 2 , also known as PIP 2 ) lipid molecules. The TRIC-B1/B2 proteins and PIP 2 assemble into a symmetrical homotrimeric complex. Each monomer contains an hourglass-shaped hydrophilic pore contained within a seven-transmembrane-helix domain. Structural and functional analyses unravel the central role of PIP 2 in stabilizing the cytoplasmic gate of the ion permeation pathway and reveal a marked Ca 2+ -induced conformational change in a cytoplasmic loop above the gate. A mechanistic model has been proposed to account for the complex gating mechanism of TRIC channels.

The voltage-dependent anion-selective channel (VDAC) is an intrinsic β-barrel membrane protein located within the mitochondrial outer membrane where it serves as a pore, connecting the mitochondria to the cytosol. The high-resolution structures of both the human and murine VDACs have been resolved by X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) in 2008. However, the structural data are not completely in line with the findings that were obtained after decades of research on biochemical and functional analysis of VDAC. This discrepancy may be related to the fact that structural biology studies of membrane proteins reveal specific static conformations that may not necessarily represent the physiological state. For example, overexpression of membrane proteins in bacterial inclusion bodies or simply the extraction from the native lipid environment using harsh purification methods (i.e. chaotropic agents) can disturb the physiological conformations and the supramolecular assemblies. To address these potential issues, we have developed a method, allowing rapid one step purification of endogenous VDAC expressed in the native mitochondrial membrane without overexpression of recombinant protein or usage of harsh chaotropic extraction procedures. Using the Saccharomyces cerevisiae isoform 1 of VDAC as a model, this method yields efficient purification, preserving VDAC in a more physiological, native state following extraction from mitochondria. Single particle analysis using transmission electron microscopy (TEM) demonstrated conservation of oligomeric assembly after purification. Maintenance of the native state was evaluated using functional assessment that involves an ATP-binding assay by micro-scale thermophoresis (MST). Using this approach, we were able to determine for the first time the apparent KD for ATP of 1.2 mM., (Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.

It is estimated that more than 170 million people are infected with hepatitis C virus (HCV) worldwide. Clinical trials have demonstrated that, for the first time in human history, the potential exists to eradicate a chronic viral disease using combination therapies that contain only direct-acting antiviral agents. HCV non-structural protein 5A (NS5A) is a multifunctional protein required for several stages of the virus replication cycle. NS5A replication complex inhibitors, exemplified by daclatasvir (DCV; also known as BMS-790052 and Daklinza), belong to the most potent class of direct-acting anti-HCV agents described so far, with in vitro activity in the picomolar (pM) to low nanomolar (nM) range. The potency observed in vitro has translated into clinical efficacy, with HCV RNA declining by ~3-4 log10 in infected patients after administration of single oral doses of DCV. Understanding the exceptional potency of DCV was a key objective of this study. Here we show that although DCV and an NS5A inhibitor analogue (Syn-395) are inactive against certain NS5A resistance variants, combinations of the pair enhance DCV potency by >1,000-fold, restoring activity to the pM range. This synergistic effect was validated in vivo using an HCV-infected chimaeric mouse model. The cooperative interaction of a pair of compounds suggests that NS5A protein molecules communicate with each other: one inhibitor binds to resistant NS5A, causing a conformational change that is transmitted to adjacent NS5As, resensitizing resistant NS5A so that the second inhibitor can act to restore inhibition. This unprecedented synergistic anti-HCV activity also enhances the resistance barrier of DCV, providing additional options for HCV combination therapy and new insight into the role of NS5A in the HCV replication cycle.

Extracellular superoxide dismutase (EC-SOD) is expressed by both macrophages and neutrophils and is known to influence the inflammatory response. Upon activation, neutrophils generate hypochlorous acid (HOCl) and secrete proteases to combat invading microorganisms. This produces a hostile environment in which enzymatic activity in general is challenged. In this study, we show that EC-SOD exposed to physiologically relevant concentrations of HOCl remains enzymatically active and retains the heparin-binding capacity, although HOCl exposure established oxidative modification of the N-terminal region (Met32) and the formation of an intermolecular cross-link in a fraction of the molecules. The cross-linking was also induced by activated neutrophils. Moreover, we show that the neutrophil-derived proteases human neutrophil elastase and cathepsin G cleaved the N-terminal region of EC-SOD irrespective of HOCl oxidation. Although the cleavage by elastase did not affect the quaternary structure, the cleavage by cathepsin G dissociated the molecule to produce EC-SOD monomers. The present data suggest that EC-SOD is stable and active at the site of inflammation and that neutrophils have the capacity to modulate the biodistribution of the protein by generating EC-SOD monomers that can diffuse into tissue., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The mitochondrial enzyme glutaminase C (GAC) catalyzes the hydrolysis of glutamine to glutamate plus ammonia, a key step in the metabolism of glutamine by cancer cells. Recently, we discovered a class of allosteric inhibitors of GAC that inhibit cancer cell growth without affecting their normal cellular counterparts, with the lead compound being the bromo-benzophenanthridinone 968. Here, we take advantage of mouse embryonic fibroblasts transformed by oncogenic Dbl, which hyperactivates Rho GTPases, together with (13)C-labeled glutamine and stable-isotope tracing methods, to establish that 968 selectively blocks the enhancement in glutaminolysis necessary for satisfying the glutamine addiction of cancer cells. We then determine how 968 inhibits the catalytic activity of GAC. First, we developed a FRET assay to examine the effects of 968 on the ability of GAC to undergo the dimer-to-tetramer transition necessary for enzyme activation. We next demonstrate how the fluorescence of a reporter group attached to GAC provides a direct read-out of the binding of 968 and related compounds to the enzyme. By combining these fluorescence assays with newly developed GAC mutants trapped in either the monomeric or dimeric state, we show that 968 has the highest affinity for monomeric GAC and that the dose-dependent binding of 968 to GAC monomers directly matches its dose-dependent inhibition of enzyme activity and cellular transformation. Together, these findings highlight the requirement of tetramer formation as the mechanism of GAC activation and shed new light on how a distinct class of allosteric GAC inhibitors impacts the metabolic program of transformed cells.

The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Alcohol dehydrogenase (ADH, EC 1.1.1.1) plays important roles in the metabolism of alcohols and aldehydes. They are often subjected to conformational changes that are critical for the enzymatic activity and have received intensive investigation for horse liver ADH. However, for the large plant ADH members, little is known regarding both the conformational change and its relationship to catalytic activity as plant ADH structures were rarely available. Here we describe three Arabidopsis ADH conformations obtained from two crystals, the apo crystal that was free of ligand, and the complex crystal that was with NAD. The NAD-complexed crystal yielded two different structural forms for the two subunits, one was occupied by the coenzyme, and the other was free and open. Structural comparisons demonstrate that the occupied subunit is in a closed conformation while the free subunit is fully open, and the apo structure in intermediate. Though all the forms have an overall fold similar to that of horse and human ADHs, the catalytic domain has an over 10° rotation. Additionally, unlike horse liver ADH, the loop (295-302aa) adopts different conformation. It does not rearrange upon the binding of the coenzyme norVal297 side chain experiences a flipping. Instead it always remains in the active site. His48 plays a switching role in the structure. Its imidazole ring has to swim away from the binding site to permit NAD binding. These together with the large differences in the substrate binding pocket, as well as in the proton relay system demonstrate that AtADH adopts a different catalysis mechanism from horse liver ADH., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

Effect of trimethylamine N-oxide (TMAO), well-known osmolyte, widely used to imitate crowded intracellular conditions, on the quaternary structure of recombinant human small heat shock protein HspB6 (Hsp20) was analyzed by means of size-exclusion chromatography, chemical crosslinking and analytical ultracentrifugation. Consistent with previous reports, in the absence of TMAO unphosphorylated, pseudophosphorylated (S16D mutant) and phosphorylated HspB6 form only small oligomers (presumably dimers). Addition of TMAO to unphosphorylated HspB6 leads to formation of different large oligomers being in equilibrium with dimers. Pseudophosphorylation (S16D mutation) or phosphorylation partially or completely prevent TMAO-induced oligomerization of HspB6. Pseudophosphorylation affects bis-ANS binding suggesting decreased hydrophobicity of HspB6. According to size-exclusion chromatography, TMAO-induced changes of HspB6 oligomerization result in its altered interaction with HspB1 and this effect can be reversed by HspB6 phosphorylation. It is concluded that under conditions of molecular crowding, characteristic for intracellular environment, HspB6 undergoes reversible changes of its oligomeric state which can affect its physiologically important properties and can be delicately regulated by phosphorylation., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

Binding to the primary receptor, CD4, triggers conformational changes in the metastable HIV-1 envelope glycoprotein (Env) trimer ((gp120-gp41)3) that are important for virus entry into host cells. These changes include an 'opening' of the trimer, creation of a binding site for the CCR5 co-receptor and formation and/or exposure of a gp41 coiled coil. Here we identify a new compound, 18A (1), that specifically inhibits the entry of a wide range of HIV-1 isolates. 18A does not interfere with CD4 or CCR5 binding, but it inhibits the CD4-induced disruption of quaternary structures at the trimer apex and the exposure of the gp41 HR1 coiled coil. Analysis of HIV-1 variants with increased or reduced sensitivity to 18A suggests that the inhibitor can distinguish distinct conformational states of gp120 in the unliganded Env trimer. The broad-range activity and observed hypersensitivity of resistant mutants to antibody neutralization support further investigation of 18A.

Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs are considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast. This is most evident in the treatment of lysosomal storage disorders, cystic fibrosis, and nephrogenic diabetes insipidus, for which proof of principle in humans has been demonstrated., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The thermodynamics of formation of the insulin hexamer, which is stabilized by two Zn(2+) ions, were quantified by isothermal titration calorimetry (ITC). Because the insulin monomer is unstable to aggregation (fibrillation) during ITC measurements, an original method involving EDTA chelation of Zn(2+) from the hexamer was employed. The two metal ions are chelated sequentially, reflecting stepwise Zn(2+) binding and stabilization of the quaternary structure. Analysis of the ITC data reveals that two to three H(+) bind to the hexamer upon its formation at pH 7.4, which is both enthalpically and entropically favored. The former is due to Zn(2+) coordination to His residues from three subunits, and the latter is associated with desolvation that accompanies the protonation and the packing of the subunits in the hexamer.

Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands against β amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter β amyloid (Aβ) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aβ1-42 directly or microglial (BV-2 cells) conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ(9)-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55 ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aβ1-42. Aβ1-42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aβ-evoked SH-SY5Y cell viability, whereas JWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aβ fibrils and aggregates, there was no clear correlation between effects on Aβ morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.

The effect of non-denaturing concentrations of three different organic solvents, formamide, acetone and isopropanol, on the structure of haloalkane dehalogenases DhaA, LinB, and DbjA at the protein-solvent interface was studied using molecular dynamics simulations. Analysis of B-factors revealed that the presence of a given organic solvent mainly affects the dynamical behavior of the specificity-determining cap domain, with the exception of DbjA in acetone. Orientation of organic solvent molecules on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydrophilic surface patches, and the simulations suggest that the behavior of studied organic solvents in the vicinity of hyrophobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied haloalkane dehalogenases and provided an opportunity to explore effects of organic solvents on the quaternary structure. Penetration and trapping of organic solvents in the network of interactions between both monomers depends on the physico-chemical properties of the organic solvents. Consequently, both monomers of this enzyme oscillate differently in different organic solvents. With the exception of LinB in acetone, the structures of studied enzymes were stabilized in water-miscible organic solvents.

FtsZ is a bacterial cytoskeletal protein involved in cell division. It forms a ringlike structure that attaches to the membrane to complete bacterial division. It binds and hydrolyzes GTP, assembling into polymers in a GTP-dependent manner. To test how the orientation of the monomers affects the curvature of the filaments on a surface, we performed site-directed mutagenesis on the E. coli FtsZ protein to insert cysteine residues at lateral locations to orient FtsZ on planar lipid bilayers. The E93C and S255C mutants were overproduced, purified, and found to be functionally active in solution, as well as being capable of sustaining cell division in vivo in complementation assays. Atomic force microscopy was used to observe the shape of the filament fibers formed on the surface. The FtsZ mutants were covalently linked to the lipids and could be polymerized on the bilayer surface in the presence of GTP. Unexpectedly, both mutants assembled into straight structures. E93C formed a well-defined lattice with monomers interacting at 60° and 120° angles, whereas S255C formed a more open array of straight thicker filament aggregates. These results indicate that filament curvature and bending are not fixed and that they can be modulated by the orientation of the monomers with respect to the membrane surface. As filament curvature has been associated with the force generation mechanism, these results point to a possible role of filament membrane attachment in lateral association and curvature, elements currently identified as relevant for force generation.

Endothelial-myocardial interactions may be critically important for ischemia/reperfusion injury. Tetrahydrobiopterin (BH4) is a required cofactor for nitric oxide (NO) production by endothelial NO synthase (eNOS). Hyperglycemia (HG) leads to significant increases in oxidative stress, oxidizing BH4 to enzymatically incompetent dihydrobiopterin. How alterations in endothelial BH4 content impact myocardial ischemia/reperfusion injury remains elusive. The aim of this study was to examine the effect of endothelial-myocardial interaction on ischemia/reperfusion injury, with an emphasis on the role of endothelial BH4 content. Langendorff-perfused mouse hearts were treated by triton X-100 to produce endothelial dysfunction and subsequently subjected to 30 min of ischemia followed by 2 h of reperfusion. The recovery of left ventricular systolic and diastolic function during reperfusion was impaired in triton X-100 treated hearts compared with vehicle-treated hearts. Cardiomyocytes (CMs) were co-cultured with endothelial cells (ECs) and subsequently subjected to 2 h of hypoxia followed by 2 h of reoxygenation. Addition of ECs to CMs at a ratio of 1∶3 significantly increased NO production and decreased lactate dehydrogenase activity compared with CMs alone. This EC-derived protection was abolished by HG. The addition of 100 µM sepiapterin (a BH4 precursor) or overexpression of GTP cyclohydrolase 1 (the rate-limiting enzyme for BH4 biosynthesis) in ECs by gene trasfer enhanced endothelial BH4 levels, the ratio of eNOS dimer/monomer, eNOS phosphorylation, and NO production and decreased lactate dehydrogenase activity in the presence of HG. These results demonstrate that increased BH4 content in ECs by either pharmacological or genetic approaches reduces myocardial damage during hypoxia/reoxygenation in the presence of HG. Maintaining sufficient endothelial BH4 is crucial for cardioprotection against hypoxia/reoxygenation injury.

Spinocerebellar ataxia type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases all characterized by the presence of intraneuronal inclusions that contain aggregated protein. Aggregation of ataxin-3, the causative protein of SCA3, has been well characterized in vitro, with both pathogenic and non-pathogenic length ataxin-3 undergoing fibrillogenesis. However, only ataxin-3 containing an expanded polyQ tract leads to SCA3. Therefore other cellular factors, not present in previous in vitro studies, may modulate aggregation during disease. The interactions between fibrillar species and cell membranes have been characterized in a number of amyloid diseases, including Huntington's Disease, and these interactions affect aggregation and toxicity. We have characterized the effects of the membrane mimetic sodium dodecyl sulfate (SDS) on ataxin-3 structure and aggregation, to show that both micellar and non-micellar SDS have differing effects on the two stages of ataxin-3 aggregation. We also demonstrate that fibrillar ataxin-3 binds phospholipids, in particular phosphorylated phosphotidylinositols. These results highlight the effect of intracellular factors on the ataxin-3 misfolding landscape and their implications in SCA3 and polyQ diseases in general are discussed.

There is a consensus that oxidative stress promotes neurodegeneration and may be linked to plaque formation. α-Synuclein is the main component of neurodegenerative plaques. We have found that α-synuclein binds strongly to the enzyme phospholipase Cβ1 (PLCβ1) in vitro and in cells affecting both its G protein activation and its degradation. Because PLCβ1 binds to α-synuclein in cells, we tested whether decreasing its level would promote α-synuclein aggregation and whether overproducing PLCβ1 would inhibit aggregation. By imaging fluorescent α-synuclein in living HEK293, PC12, and SK-H-SH cells, we find that α-synuclein aggregation is directly related to the level of PLCβ1. Importantly, we found that oxidative stress does not affect the cellular levels of α-synuclein but results in the down-regulation of PLCβ1 thereby promoting α-synuclein aggregation. A peptide that mimics part of the α-synuclein binding site to PLCβ prevents aggregation. Our studies indicate that PLCβ1 can reduce cell damage under oxidative stress and offers a potential site that might be exploited to prevent α-synuclein aggregation.

The development of new therapeutics for the treatment of neurodegenerative pathophysiologies currently stands at a crossroads. This presents an opportunity to transition future drug discovery efforts to target disease modification, an area in which much still remains unknown. In this Perspective we examine recent progress in the areas of neurodegenerative drug discovery, focusing on some of the most common targets and mechanisms: N-methyl-d-aspartic acid (NMDA) receptors, voltage gated calcium channels (VGCCs), neuronal nitric oxide synthase (nNOS), oxidative stress from reactive oxygen species, and protein aggregation. These represent the key players identified in neurodegeneration and are part of a complex, intertwined signaling cascade. The synergistic delivery of two or more compounds directed against these targets, along with the design of small molecules with multiple modes of action, should be explored in pursuit of more effective clinical treatments for neurodegenerative diseases.

Depolymerization of xylan, a major fraction of lignocellulosic biomass, releases xylose which can be converted into transportation fuels and chemical feedstocks. A requisite enzyme for the breakdown of xylan is β-xylosidase. A gene encoding the 324-amino acid β-xylosidase, RS223-BX, was cloned from an anaerobic mixed microbial culture. This glycoside hydrolase belongs to family 43. Unlike other GH43 enzymes, RS223-BX can be strongly activated by exogenously supplied Ca(2+), Co(2+), Fe(2+), Mg(2+), Mn(2+) and Ni(2+) (e.g., 28-fold by Mg(2+)) and it is inhibited by Cu(2+) or Zn(2+). Sedimentation equilibrium centrifugation experiments indicated that the divalent metal cations mediate multimerization of the enzyme from a dimeric to a tetrameric state, which have equal catalytic activity on an active-site basis. Compared to the determined active sites of other GH43 β-xylosidases, the predicted active site of RS223-BX contains two additional amino acids with carboxylated side chains that provide potential sites for divalent metal cations to reside. Thus, the divalent metal cations likely occupy the active site and participate in the catalytic mechanism. RS223-BX accepts as substrate xylobiose, arabinobiose, 4-nitrophenyl-β-D-xylopyranoside, and 4-nitrophenyl-α-L-arabinofuranoside. Additionally, the enzyme has good pH and temperature stabilities and a large K(i) for D-glucose (1.3 M), favorable properties for performance in saccharification reactors., (Published by Elsevier Inc.)

Hemolytic lectin CEL-III isolated from the sea cucumber Cucumaria echinata forms transmembrane pores by self-oligomerization in target cell membranes. It also formed soluble oligomers in aqueous solution upon binding with specific carbohydrates under conditions of high pH and a high salt concentration. The size of the soluble CEL-III oligomers decreased when treated with detergents such as Triton X-100 and SDS. Small-angle X-ray scattering measurements suggested that the dissociated unit of the oligomer was a tightly associated CEL-III heptamer. Without detergents in solution, these heptamers further assembled into larger 21mer oligomers, comprising three heptamers held together by relatively weak hydrophobic interactions.

Purpose: We investigated the in vitro amyloid aggregation ability of TGFBI (transforming growth factor beta-induced) mutants causing corneal dystrophies (CDs)., Methods: Peripheral blood samples were collected from 42 unrelated Chinese CD patients and 185 healthy subjects for mutation screening in all TGFBI coding exons and flanking introns. The expression vector pCMV6_TGFBI containing wild-type, Arg-124, or Arg-555 mutations was transfected to HEK293 cells. Cell-free media was incubated with amyloid-beta (Aβ) (1-40) peptides with or without a chemical osmolyte, trimethylamine N-oxide (TMAO), for different time intervals. After ultracentrifugation, protein aggregates were analyzed by denatured gel electrophoresis. The effect of TMAO on chemical and morphological properties of Aβ aggregation was examined., Results: TGFBI sequencing analysis showed c.Arg124Cys in all 6 lattice CD patients, c.Arg555Glu in all 11 granular CD type 1 patients, and c.Arg124His in 22 of 25 granular CD type 2 patients. Double heterozygosity (c.307-308delCT and c.Arg124His) was detected in one GCD2 patient. After transfection, cell-free media containing Arg-124 TGFBIp led to Aβ aggregation within 12 hours, whereas wild-type and Arg-555 mutant displayed aggregation after 24 hours. Western blot and Congo red binding assays showed that TMAO dose-dependently suppressed Arg-124-induced Aβ aggregation. Transmission electron microscopy showed that TMAO reduced the fibrillar aggregates caused by Aβ and c.124R > H mutated TGFBIp., Conclusions: TGFBI sequence heterogeneity was observed in Chinese CD patients. TMAO reduced amyloid aggregation caused by Arg-124 mutants, which suggests a potential chemical-based treatment for CDs.

Alzheimer's disease is a debilitating neurodegenerative disorder whose pathology has been linked to the aggregation and deposition of the amyloid β-peptide (Aβ) in neural tissue. A truly effective therapeutic agent remains elusive, and attention has recently turned to the use of natural products as effective antiaggregation compounds, directly targeting Aβ. Although a wealth of in vitro and in vivo evidence suggests these compounds or their derivatives might be beneficial, a detailed understanding of the mechanism by which they act remains largely unknown. Using atomistic, explicit-solvent molecular dynamics simulations, we have investigated the association of the flavonoid morin with Aβ monomers and dimers. Through 90 simulations totaling 23.65 μs, we found that treatment of Aβ peptides with morin largely does not affect secondary structure content, unless a large molar excess of morin is present. However, in simulations of Aβ monomers and dimers, morin affected the tertiary and quaternary structure of Aβ, even at low concentrations that have been used experimentally. Thus it appears that despite the inability of morin to fully block Aβ aggregation or β-strand formation, we observe structures with altered tertiary and quaternary interactions, which may represent "off-pathway" aggregates that have been proposed previously. The simulations presented here add important new details to the mechanism of these processes.

S-layers are regular two-dimensional semipermeable protein layers that constitute a major cell-wall component in archaea and many bacteria. The nanoscale repeat structure of the S-layer lattices and their self-assembly from S-layer proteins (SLPs) have sparked interest in their use as patterning and display scaffolds for a range of nano-biotechnological applications. Despite their biological abundance and the technological interest in them, structural information about SLPs is limited to truncated and assembly-negative proteins. Here we report the X-ray structure of the SbsB SLP of Geobacillus stearothermophilus PV72/p2 by the use of nanobody-aided crystallization. SbsB consists of a seven-domain protein, formed by an amino-terminal cell-wall attachment domain and six consecutive immunoglobulin-like domains, that organize into a φ-shaped disk-like monomeric crystallization unit stabilized by interdomain Ca(2+) ion coordination. A Ca(2+)-dependent switch to the condensed SbsB quaternary structure pre-positions intermolecular contact zones and renders the protein competent for S-layer assembly. On the basis of crystal packing, chemical crosslinking data and cryo-electron microscopy projections, we present a model for the molecular organization of this SLP into a porous protein sheet inside the S-layer. The SbsB lattice represents a previously undescribed structural model for protein assemblies and may advance our understanding of SLP physiology and self-assembly, as well as the rational design of engineered higher-order structures for biotechnology.