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Alzheimer's disease (AD) is characterized by fibrillar deposits of amyloid-β (Aβ) peptides and neurofibrillary tangles of Tau proteins. Aβ peptides are composed of 37-49 residues, among which the Aβ 42 isoform is particularly toxic and aggregation-prone and is enriched in the plaques of AD brains and thus considered central to the development of AD. Therefore, disaggregation and disruption provide potential therapeutic approaches to reduce, inhibit, and even reverse Aβ aggregation. Here we capture the atomic-level details of the interactions between sigmoid Aβ 42 fibril 2MXU or 5KK3 and either natural tanshinone compounds TS1 or TS0 or negatively charged ER, proposing two unprecedented disassembly mechanisms. Natural TS1 or TS0 prefers to insert into the cavity together with part at the surface of the 2MXU to open up the mouth and twist the conformation, destroying the ordered growth of subsequent monomers along the fibril axis. For the more compact two-fold 5KK3 , attachment of TS1 or TS0 at the surface including some inserted in cavity results in the separation of the two folds. In the two sigmoid fibril systems, it is no longer applicable for the routine criteria to assess Aβ 42 fibril disassembly by introduction of these drugs, such as either reduced H-bond number, decreased β-sheet contents, or both. ER, like-charged to Aβ 42 fibril, is especially exceptional, and departs utterly from the neutral ones to disassemble Aβ 42 fibril. Besides the inapplicable routine criteria, positive binding energy between ER and Aβ 42 fibril also deviates from the hypotheses of "ligands exhibiting greater affinity for the β-amyloid peptide are effective at altering its aggregation and inhibiting cell toxicity" ( Cairo et al. , Biochemistry 2002 , 41 , 8620 - 8629 ) but results in stronger disassembly effect on the two kinds of sigmoid Aβ 42 fibrils than neutral TS0 or TS1. The disassembly power of charged ER molecules derives from its stronger deformation ability to the conformation of Aβ 42 fibril than the neutral ones, twisting the one-fold 2MXU into tapered-shape and separating two-fold 5KK3 in two parts further, which is in great agreement with experimental observations ( Irwin et al. Biomacromolecules 2013 , 14 ( 1 ), 264 - 274 ). The unusual disassembly mechanisms fill the gaps and offer an alternative direction in engineering new inhibitors to treat AD.

EGFR is a receptor tyrosine kinase that plays a critical role in cell proliferation, differentiation, survival and migration. Its activating ligand, EGF, has long been believed to stabilize the EGFR dimer. Two research studies aimed at quantitative measurements of EGFR dimerization, however, have led to contradicting conclusions and have questioned this view. Given the controversy, here we sought to measure the dimerization of EGFR in the absence and in the presence of saturating EGF concentrations, and to tease out the effect of ligand on dimer stability, using a FRET-based quantitative method. Our measurements show that the dissociation constant is decreased ~150 times due to ligand binding, indicative of significant dimer stabilization. In addition, our measurements demonstrate that EGF binding induces a conformational change in the EGFR dimer., (Copyright © 2019. Published by Elsevier B.V.)

Heat shock protein 90 (HSP90) is a promising target for treatment of cancer, and inhibitor bindings can generate efficient suppression on tumor in multiple ways. In this work, 140-ns molecular dynamics simulations were performed on six systems. Principal component analysis was subsequently carried out to explore the conformational diversity of HSP90. The results suggest that inhibitor bindings induce large conformational changes of HSP90, which tends to enlarge the volume of the binding pocket to facilitate the entrance of inhibitors. Hierarchical clustering analyses, the calculation of the energy contribution of each atom, and the analyses of hydrogen-bonding interactions were performed. The results indicate that 20 residues in group A of the hierarchical tree are responsible for major contributions, and van der Waals interactions as well as hydrogen-bonding interactions between important residues in HSP90 and key regions of inhibitors are the main force for promoting inhibitor bindings. We expect that this work can provide useful theoretical information for development of efficient inhibitors targeting HSP90., (© 2019 John Wiley & Sons A/S.)

Iodide ions (I - ) are an essential dietary mineral, and crucial for mental and physical development, fertility and thyroid function. I - is also a high affinity substrate for the heme enzyme myeloperoxidase (MPO), which is involved in bacterial cell killing during the immune response, and also host tissue damage during inflammation. In the presence of H 2 O 2 and Cl - , MPO generates the powerful oxidant hypochlorous acid (HOCl), with excessive formation of this species linked to multiple inflammatory diseases. In this study, we have examined the hypothesis that elevated levels of I - would decrease HOCl formation and thereby protein damage induced by a MPO/Cl - /H 2 O 2 system, by acting as a competitive substrate. The presence of increasing I - concentrations (0.1-10 μM; i.e. within the range readily achievable by oral supplementation in humans), decreased damage to both model proteins and extracellular matrix components as assessed by gross structural changes (SDS-PAGE), antibody recognition of parent and modified protein epitopes (ELISA), and quantification of both parent amino acid loss (UPLC) and formation of the HOCl-biomarker 3-chlorotyrosine (LC-MS) (reduced by ca. 50% at 10 μM I - ). Elevated levels of I - ( > 1 μM) also protected against functional changes as assessed by a decreased loss of adhesion (eg. 40% vs. < 22% with >1 μM I - ) of primary human coronary artery endothelial cells (HCAECs), to MPO-modified human plasma fibronectin. These data indicate that low micromolar concentrations of I - , which can be readily achieved in humans and are readily tolerated, may afford protection against cell and tissue damage induced by MPO., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The bacterial fatty acid pathway is essential for membrane synthesis and a range of other metabolic and cellular functions. The β-ketoacyl-ACP synthases carry out the initial elongation reaction of this pathway, utilizing acetyl-CoA as a primer to elongate malonyl-ACP by two carbons, and subsequent elongation of the fatty acyl-ACP substrate by two carbons. Here we describe the structures of the β-ketoacyl-ACP synthase I from Brucella melitensis in complex with platencin, 7-hydroxycoumarin, and (5-thiophen-2-ylisoxazol-3-yl)methanol. The enzyme is a dimer and based on structural and sequence conservation, harbors the same active site configuration as other β-ketoacyl-ACP synthases. The platencin binding site overlaps with the fatty acyl compound supplied by ACP, while 7-hydroxyl-coumarin and (5-thiophen-2-ylisoxazol-3-yl)methanol bind at the secondary fatty acyl binding site. These high-resolution structures, ranging between 1.25 and 1.70 å resolution, provide a basis for in silico inhibitor screening and optimization, and can aid in rational drug design by revealing the high-resolution binding interfaces of molecules at the malonyl-ACP and acyl-ACP active sites., (© 2019 Wiley Periodicals, Inc.)

The X-ray structure of lysozyme from bacteriophage lambda (λ lysozyme) in complex with the inhibitor hexa-N-acetylchitohexaose (NAG6) (PDB: 3D3D) has been reported previously showing sugar units from two molecules of NAG6 bound in the active site. One NAG6 is bound with four sugar units in the ABCD sites and the other with two sugar units in the E'F' sites potentially representing the cleavage reaction products; each NAG6 cross links two neighboring λ lysozyme molecules. Here we use NMR and MD simulations to study the interaction of λ lysozyme with the inhibitors NAG4 and NAG6 in solution. This allows us to study the interactions within the complex prior to cleavage of the polysaccharide. 1 H N and 15 N chemical shifts of λ lysozyme resonances were followed during NAG4/NAG6 titrations. The chemical shift changes were similar in the two titrations, consistent with sugars binding to the cleft between the upper and lower domains; the NMR data show no evidence for simultaneous binding of a NAG6 to two λ lysozyme molecules. Six 150 ns MD simulations of λ lysozyme in complex with NAG4 or NAG6 were performed starting from different conformations. The simulations with both NAG4 and NAG6 show stable binding of sugars across the D/E active site providing low energy models for the enzyme-inhibitor complexes. The MD simulations identify different binding subsites for the 5th and 6th sugars consistent with the NMR data. The structural information gained from the NMR experiments and MD simulations have been used to model the enzyme-peptidoglycan complex., (© 2019 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals, Inc.)

Seven ascidiacyclamide [cyclo(-Ile-oxazoline-d-Val-thiazole-) 2 ] (ASC) analogues incorporating the β-amino acids βIle, βoxazoline, and/or d-βVal were synthesized. We then investigated the effects of the position and number of incorporated β-amino acids on the structure, cytotoxicity, and copper binding by these seven analogues. The structural analyses revealed that both βIle and d-βVal favor a gauche-type θ torsion angles, while βoxazoline favors a trans-type θ torsion angle. Expansion of the macrocycle by incorporation of βIle or d-βVal readily induced molecular folding. On the other hand, the incorporation of two βoxazoline residues strongly extended the peptide conformation, and the incorporation of one was sufficient for the moderate restriction important for conformational equilibrium and cytotoxicity. Despite expansion of the macrocycles, the structure-cytotoxicity relationships were largely maintained. In studies of complexation of the analogues with Cu (II) ion, the position and number of incorporated β-amino acids had a large impact on the structure of the metal complex and may contribute to its stabilization., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

Experiments face challenges in the analysis of intrinsically disordered proteins in solution due to fast conformational changes and enhanced aggregation propensity. Computational studies complement experiments, being widely used in the analyses of intrinsically disordered proteins, especially those positioned at the centers of neurodegenerative diseases. However, recent investigations - including our own - revealed that computer simulations face significant challenges and limitations themselves. In this review, we introduced and discussed some of the scientific challenges and limitations of computational studies conducted on intrinsically disordered proteins. We also outlined the importance of future developments in the areas of computational chemistry and computational physics that would be needed for generating more accurate data for intrinsically disordered proteins from computer simulations. Additional theoretical strategies that can be developed are discussed herein., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C 12 E 8 ). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C 12 E 8 leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C 12 E 8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C 12 E 8 recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C 12 E 8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aurora kinases are essential enzymes for the control of cell cycle. The specific role of aurora kinase A (AURKA) is the regulation of spindle assembly and stability by promoting centrosome maturation and separation. Because AURKA is an essential protein for the normal occurrence of the cycle, mutations and deregulations in the activities of this protein are associated with several cancers. The kinase activity of AURKA is controlled by autocatalytic phosphorylation, which is facilitated after binding to a regulator protein, the Target Protein for Xenopuskinesin-like protein 2 (TPX2)., Scope of Review: This review highlights the physiological and pathophysiological properties of AURKA, the structure of the AURKA/TPX2 complex and the main structural features that can be explored for the design of selective AURKA inhibitors., Major Conclusions: The design of selective AURKA inhibitors remains as a challenge as most of the currently available inhibitors target only the ATP binding cleft and are nonselective among kinases. However, by exploring the inactive form of the kinase, researchers get access to an adjacent hydrophobic pocket, allowing the design of more selective inhibitors. Additionally, the possibility of designing potent allosteric AURKA inhibitors look very promising from the clinical perspective, since it tends to yield the most selective class of compounds., General Significance: Herein we detailed the binding modes of the most selective AURKA inhibitors currently reported. We believe this will aid researchers in defining the structural patterns necessary for selective AURKA inhibition, guiding the design of more potent compounds to be therapeutically explored in cancer patients., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The most common dementia worldwide, Alzheimer's disease is often diagnosed via biomarkers in cerebrospinal fluid, including reduced levels of Aβ1-42, and increases in total tau and phosphorylated tau-181. Here we describe results of a Phase 2a study of a promising new drug candidate that significantly reversed all measured biomarkers of Alzheimer's disease, neurodegeneration and neuroinflammation. PTI-125 is an oral small molecule drug candidate that binds and reverses an altered conformation of the scaffolding protein filamin A found in Alzheimer's disease brain. Altered filamin A links to the α7-nicotinic acetylcholine receptor to allow Aβ42's toxic signaling through this receptor to hyperphosphorylate tau. Altered filamin A also links to toll-like receptor 4 to enable Aβ-induced persistent activation of this receptor and inflammatory cytokine release. Restoring the native shape of filamin A prevents or reverses filamin A's linkages to the α7-nicotinic acetylcholine receptor and toll-like receptor 4, thereby blocking Aβ42's activation of these receptors. The result is reduced tau hyperphosphorylation and neuroinflammation, with multiple functional improvements demonstrated in transgenic mice and postmortem Alzheimer's disease brain., Objectives: Safety, pharmacokinetics, and cerebrospinal fluid and plasma biomarkers were assessed following treatment with PTI-125 for 28 days. Target engagement and mechanism of action were assessed in patient lymphocytes by measuring 1) the reversal of filamin A's altered conformation, 2) linkages of filamin A with α7-nicotinic acetylcholine receptor or toll-like receptor 4, and 3) levels of Aβ42 bound to α7-nicotinic acetylcholine receptor or CD14, the co-receptor for toll-like receptor 4., Design: This was a first-in-patient, open-label Phase 2a safety, pharmacokinetics and biomarker study., Setting: Five clinical trial sites in the U.S. under an Investigational New Drug application., Participants: This study included 13 mild-to-moderate Alzheimer's disease patients, age 50-85, Mini Mental State Exam ≥16 and ≤24 with a cerebrospinal fluid total tau/Aβ42 ratio ≥0.30., Intervention: PTI-125 oral tablets (100 mg) were administered twice daily for 28 consecutive days., Measurements: Safety was assessed by electrocardiograms, clinical laboratory analyses and adverse event monitoring. Plasma levels of PTI-125 were measured in blood samples taken over 12 h after the first and last doses; cerebrospinal fluid levels were measured after the last dose. Commercial enzyme linked immunosorbent assays assessed levels of biomarkers of Alzheimer's disease in cerebrospinal fluid and plasma before and after treatment with PTI-125. The study measured biomarkers of pathology (pT181 tau, total tau and Aβ42), neurodegeneration (neurofilament light chain and neurogranin) and neuroinflammation (YKL-40, interleukin-6, interleukin-1β and tumor necrosis factor α). Plasma levels of phosphorylated and nitrated tau were assessed by immunoprecipitation of tau followed by immunoblotting of three different phospho-epitopes elevated in AD (pT181-tau, pS202-tau and pT231-tau) and nY29-tau. Changes in conformation of filamin A in lymphocytes were measured by isoelectric focusing point. Filamin A linkages to α7-nicotinic acetylcholine receptor and toll-like receptor 4 were assessed by immunoblot detection of α7-nicotinic acetylcholine receptor and toll-like receptor 4 in anti-filamin A immunoprecipitates from lymphocytes. Aβ42 complexed with α7-nicotinic acetylcholine receptor or CD14 in lymphocytes was also measured by co-immunoprecipitation. The trial did not measure cognition., Results: Consistent with the drug's mechanism of action and preclinical data, PTI-125 reduced cerebrospinal fluid biomarkers of Alzheimer's disease pathology, neurodegeneration and neuroinflammation from baseline to Day 28. All patients showed a biomarker response to PTI-125. Total tau, neurogranin, and neurofilament light chain decreased by 20%, 32% and 22%, respectively. Phospho-tau (pT181) decreased 34%, evidence that PTI-125 suppresses tau hyperphosphorylation induced by Aβ42's signaling through α7-nicotinic acetylcholine receptor. Cerebrospinal fluid biomarkers of neuroinflammation (YKL-40 and inflammatory cytokines) decreased by 5-14%. Biomarker effects were similar in plasma. Aβ42 increased slightly - a desirable result because low Aβ42 indicates Alzheimer's disease. This increase is consistent with PTI-125's 1,000-fold reduction of Aβ42's femtomolar binding affinity to α7-nicotinic acetylcholine receptor. Biomarker reductions were at least p ≤ 0.001 by paired t test. Target engagement was shown in lymphocytes by a shift in filamin A's conformation from aberrant to native: 93% was aberrant on Day 1 vs. 40% on Day 28. As a result, filamin A linkages with α7-nicotinic acetylcholine receptor and toll-like receptor 4, and Aβ42 complexes with α7-nicotinic acetylcholine receptor and CD14, were all significantly reduced by PTI-125. PTI-125 was safe and well-tolerated in all patients. Plasma half-life was 4.5 h and approximately 30% drug accumulation was observed on Day 28 vs. Day 1., Conclusions: PTI-125 significantly reduced biomarkers of Alzheimer's disease pathology, neurodegeneration, and neuroinflammation in both cerebrospinal fluid and plasma. All patients responded to treatment. The magnitude and consistency of reductions in established, objective biomarkers imply that PTI-125 treatment counteracted disease processes and reduced the rate of neurodegeneration. Based on encouraging biomarker data and safety profile, approximately 60 patients with mild-to-moderate AD are currently being enrolled in a Phase 2b randomized, placebo-controlled confirmatory study to assess the safety, tolerability and efficacy of PTI-125., Competing Interests: LHB, CAC, RB and NF are employees of Cassava Sciences, Inc. HYW and MRM are science advisors to Cassava Sciences, Inc. ELB and BN are employees of IMIC, Inc., an independent clinical site that participated in this study.

Griffithsin (Grft) is an antiviral lectin that has been shown to potently inhibit HIV-1 by binding high-mannose N-linked glycosylation sites on HIV-1 gp120. A key factor for Grft potency is glycosylation at N295 of gp120, which is directly adjacent to N332, a target glycan for an entire class of broadly neutralizing antibodies (bNAbs). Here, we unify previous work on the importance of other glycans to Grft potency against HIV-1 and Grft's role in mediating the conformational change of gp120 by mutating nearly every glycosylation site in gp120. In addition to a significant loss of Grft activity by the removal of glycosylation at N295, glycan absence at N332 or N448 was found to have moderate effects on Grft potency. Interestingly, in the absence of N295, Grft effectiveness could be improved by a mutation that results in the glycan at N448 shifting to N446, indicating that the importance of individual glycans may be related to their effect on glycosylation density. Grft's ability to alter the structure of gp120, exposing the CD4 binding site, correlated with the presence of glycosylation at N295 only in clade B strains, not clade C strains. We further demonstrate that Grft can rescue the activity of the bNAbs PGT121 and PGT126 in the event of a loss or a shift of glycosylation at N332, where the bNAbs suffer a drastic loss of potency. Despite targeting the same region, Grft in combination with PGT121 and PGT126 produced additive effects. This indicates that Grft could be an important combinational therapeutic., (Copyright © 2019 American Society for Microbiology.)

The translocator protein TSPO is in an important diagnostic and therapeutic target in a range of pathologies, including neuroinflammation and cancer. Despite the availability of several structures of TSPO homologues, our understanding of the molecular determinants that govern high-affinity interactions of TSPO with its ligands is incomplete. Here, in order to decipher the key structural elements of TSPO responsible for interactions with its ligands, we designed a panel of chimeric proteins mimicking the mammalian substrate binding site grafted onto the backbone of the Rhodobacter sphaeroides TSPO homologue, RsTSPO. One of the designed chimeric constructs, RsMouse, could be heterologously expressed and displayed improved binding affinities for the known TSPO drugs diazepam, PK11195 and NBD-FGIN-1-27. Furthermore, the chimeric protein had improved interactions with NBD-cholesterol, a fluorescent analogue of the presumed natural substrate of TSPO. Partial modifications of the transmembrane helix bundle in the chimeric construct differentially affected binding of the TSPO drugs and the natural substrates of TSPO, consistent with the presence of multiple ligand binding sites in the protein. Based on the available structures of TSPO homologues, the substrate interactions may involve a lateral opening of the protein in the TM1-3, and stabilisation of TM4-5 is important for drug-like ligand binding. These observations are consistent with our experimental results, which show that the determinants of high-affinity ligand interactions of TSPO are distinct for different classes of ligands., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Macrocycles represent a potentially vast extension of drug chemical space still largely untapped by synthetic compounds. Sampling of flexible rings is incorporated in the ICM-dock protocol. We tested the ability of ICM-dock to reproduce macrocyclic ligand-protein receptor complexes, first in a large retrospective benchmark (246 complexes), and next, in context of the D3R Grand Challenge 4 (GC4), where we modeled bound complexes and predicted activities for a series of macrocyclic BACE inhibitors. Sub-angstrom accuracy was achieved in ligand pose prediction both in cross-docking (D3R Challenge Stage 1A) and cognate (Stage 1B) setup. Stage 1B submission was top ranked by mean and average RMSDs, even though no ligand knowledge was used in our simulations on this Stage. Furthermore, we demonstrate successful receptor conformational selection in Stage 1A, aided by the enhanced '4D' multiple receptor conformation docking protocol with optimized scoring offsets. In the activity 3D QSAR modeling, predictivity of the BACE pKd model was modest, while for the second target (Cathepsin-S), leading performance was achieved. Difference in activity prediction performance between the targets is likely explained by the amount of available and relevant training data.

Mass spectrometry (MS)-based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.

Aberrant aggregation of the Aβ protein is a hallmark of Alzheimer's disease (AD), but no complete characterization of the molecular level pathogenesis has been achieved. A promising hypothesis is that dysfunction of metal ion homeostasis, and consequently, the undesired interaction of metal ions with Aβ, may be central to the development of AD. Qualitatively, most data indicate that Cu(II) induces rapid self-assembly of both Aβ40 and Aβ42 during the initial phase of the aggregation, while at longer time scales fibrillation may occur, depending on the experimental conditions. For Aβ40 and Cu(II):Aβ ≤ 1, most data imply that low concentration of Aβ40 favors nucleation and rapid fibril elongation, while high concentration of Aβ40 favors formation of amorphous aggregates. However, there are conflicting reports on this issue. For Aβ42 and Cu(II):Aβ ≤ 1, there is consensus that the lag time is extended upon addition of Cu(II). For Cu(II):Aβ > 1, the lag time is increased upon interaction with Cu(II), and in most cases fibrillation is not observed, presumably because Cu(II) occupies a second more solvent-exposed binding site, which is more prone to form metal ion-bridged species and cause rapid formation of non-fibrillar aggregates. The interesting N-terminally truncated Aβ11-40 with high affinity for Cu(II), exhibits delay of fibrillation upon addition of 0.4 eq. Cu(II). In our view, there are still problems achieving reproducible results in this field, and we provide a shortlist of some of the pitfalls. Finally, we propose a consensus model for the effects of Cu(II) on the aggregation kinetics of Aβ.

Using the D3R Grand Challenge 4 dataset containing Beta-secretase 1 (BACE) and Cathepsin S (CatS) inhibitors, we have evaluated the performance of our in-house docking workflow that involves in the first step the selection of the most suitable docking software for the system of interest based on structural and functional information available in public databases, followed by the docking of the dataset to predict the binding modes and ranking of ligands. The macrocyclic nature of the BACE ligands brought additional challenges, which were dealt with by a careful preparation of the three-dimensional input structures for ligands. This provided top-performing predictions for BACE, in contrast with CatS, where the predictions in the absence of guiding constraints provided poor results. These results highlight the importance of previous structural knowledge that is needed for correct predictions on some challenging targets. After the end of the challenge, we also carried out free energy calculations (i.e. in a non-blinded manner) for CatS using the pmx software and several force fields (AMBER, Charmm). Using knowledge-based starting pose construction allowed reaching remarkable accuracy for the CatS free energy estimates. Interestingly, we show that the use of a consensus result, by averaging the results from different force fields, increases the prediction accuracy.

Metal dyshomeostasis has long been linked to Parkinson's disease (PD), and the amyloidogenic protein α-synuclein (αS) is universally recognized as a key player in PD pathology. Structural consequences upon coordination of copper and iron to αS have gained attention due to significant dyshomeostasis of both metals in the PD brain. Protein-metal association can navigate protein folding in distinctive pathways based on the identity of the bio-metal in question. In this work, we employed photo-chemical crosslinking of unmodified proteins (PICUP) to evaluate these potential metal ion-induced structural alterations in the folding dynamics of N-terminally acetylated αS ( NAc αS) following metal coordination. Through fluorescence analysis and immunoblotting analyses following photoirradiation, we discovered that coordination of iron obstructs copper-promoted crosslinking. The absence of intra-molecular crosslinking upon iron association further supports its C-terminal coordination site and suggests a potential role for iron in mitigating nearby post-translational modification of tyrosine residues. Decreased fluorescence emission upon synergistic coordination of both copper and iron highlighted that although copper acts as a conformational promotor of NAc αS crosslinking, iron inhibits analogous conformational changes within the protein. The metal coordination preferences of NAc αS suggest that both competitive binding sites as well as dual metal coordination contribute to the changes in folding dynamics, unveiling unique structural orientations for NAc αS that have a direct and measureable influence on photoinitiated dityrosine crosslinks. Moreover, our findings have physiological implications in that iron overload, as is associated with PD-insulted brain tissue, may serve as a conformational block of copper-promoted protein oxidation.

Collagen-derived dipeptide prolyl hydroxyproline (Pro-Hyp) is involved in the proliferation and differentiation of various types of cultured cells. To elucidate the mechanism underlying Pro-Hyp actions during osteoblast differentiation, we hypothesized that proteins binding to Pro-Hyp serve to mediate cellular signaling, affecting Runx2 expression. Recently, we performed the characterization of Foxg1, that it enhances Runx2 expression in the presence of Pro-Hyp. Our findings indicate that Pro-Hyp directly binds to the Foxg1 recombinant protein, which leads to the structural alteration of the Foxg1 protein. In addition, Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp, with Pro-Hyp disrupting the interaction between Foxg1 and Runx2. Collectively, our results indicate that the Pro-Hyp bound Foxg1 alters the structured conformation of Foxg1, resulting in conformational changes that lead to dissociation from Runx2. These novel findings suggest that during osteoblast differentiation, Pro-Hyp mediates Runx2 activity though directly binding to Foxg1 and increases Runx2 expression. Abbreviations: CPT: collagen peptide; GST: Glutathione S-transferase; PAGE: Polyacrylamide gel electrophoresis; PCR: Polymerase chain reaction; prolyl hydroxyproline: Pro-Hyp.

Given the ubiquity of iodinated disinfection by-products (I-DBPs) in drinking water and their prominent toxicity, it is of vital significance to evaluate I-DBPs toxicity and explore the underlying mechanism. The toxicity of iodoacetic acid (IAA), a typical type of I-DBPs, might be linked with oxidative stress. However, it remains unknown for the response of antioxidant enzyme superoxide dismutase (SOD) in the mouse primary hepatocytes when exposed to IAA and the underlying mechanism. This study explored SOD response to IAA and the underlying mechanisms at the molecular and cellular levels. Under IAA exposure, the observed increase of SOD activity in the hepatocytes was caused by the increase of SOD production via ROS stimulation and the increase of SOD molecular activity. Molecular experiments showed that IAA binds to SOD molecule mainly via electrostatic forces with one binding site around the active site and six binding sites in the surface of protein. The binding interaction leads to the conformational changes of SOD and the disruption of protein aggregates. This work could offer basic data for the comprehensive understanding of the adverse effects of IAA and references for assessing the harmful effects of DBPs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Kinase structures in the inactive "DFG-out" state provide a wealth of druggable binding site variants. The conformational plasticity of this state can be mainly described by different conformations of binding site-forming elements such as DFG motif, A-loop, P-loop, and αC-helix. Compared to DFG-in structures, DFG-out structures are largely underrepresented in the Protein Data Bank (PDB). Thus, structure-based drug design efforts for DFG-out inhibitors may benefit from an efficient approach to generate an ensemble of DFG-out structures. Accordingly, the presented modeling pipeline systematically generates homology models of kinases in several DFG-out conformations based on a sophisticated creation of template structures that represent the major states of the flexible structural elements. Eighteen template classes were initially selected from all available kinase structures in the PDB and subsequently employed for modeling the entire kinome in different DFG-out variants by fusing individual structural elements to multiple chimeric template structures. Molecular dynamics simulations revealed that conformational transitions between the different DFG-out states generally do not occur within trajectories of a few hundred nanoseconds length. This underlines the benefits of the presented homology modeling pipeline to generate relevant conformations of "DFG-out" kinase structures for subsequent in silico screening or binding site analysis studies., (© 2019 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals, Inc.)

We previously reported that the melanocortin-1 receptor (MC1R), a key regulator of melanogenesis, regulates cell migration; however, the detailed mechanism remained unknown. Since the homo-dimerization of MC1R by four inter-subunit disulfide bonds is known to be functionally important for melanogenesis, we investigated the importance of MC1R dimerization for cell migration. Unlike the wild-type MC1R, the dimerization-defective mutant MC1R in which four critical Cys residues were replaced with Ala residues (Cys35-267-273-275Ala) significantly inhibited melanin synthesis but enhanced cell migration in human MNT-1 and A375 melanoma cells. This suggests that there may be a reverse correlation between melanin synthesis and cell migration. Interestingly, melanoma cells expressing the dimerization-defective mutant exhibited enhanced expression of the cell surface heparan sulfate proteoglycan, syndecan-2, and knockdown of syndecan-2 expression decreased the mutant-mediated cell migration. Consistently, ASIP, an antagonist of MC1R, enhanced syndecan-2 expression and cell migration and reversed the α-melanocyte-stimulating hormone (α-MSH)-mediated inhibition of syndecan-2 expression. Furthermore, α-MSH reduced the cell migration of MNT1 cells expressing wild-type MC1R but not its dimerization-defective mutant. Together, these data strongly suggest that MC1R reversely regulates melanin synthesis and migration via the conformational changes induced by dimerization., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The widespread use of aluminum in the treatment of drinking water, food, agriculture and pharmaceuticals has greatly increased the risk of human exposure to excess aluminum, which is a serious health hazard to human beings. In our previous work, serum albumin was reported to have a specific affinity for aluminum. However, the mechanism of binding of aluminum to serum albumin was unclear. In this work, the interaction between bovine serum albumin (BSA) and aluminum-maltol (Al-Mal) was studied by molecular docking and spectroscopic analysis. The results show that the combination of Al-Mal and BSA is a spontaneous endothermic reaction. The binding force is mainly related to the hydrophobic force and hydrogen bonding; when the ratio of BSA to Al-Mal was 1 : 10, the random coils of BSA increased by 47.6%. In addition, the hydrophobicity of BSA was enhanced after combining with Al-Mal. This study can provide a theoretical evidence for the binding mechanism of food-borne aluminum and serum albumin.

This study investigates the role of hydration and its relationship to the conformational equilibrium of the host molecule β-cyclodextrin. Molecular dynamics simulations indicate that the unbound β-cyclodextrin exhibits two state behavior in explicit solvent due to the opening and closing of its cavity. In implicit solvent, these transitions are not observed, and there is one dominant conformation of β-cyclodextrin with an open cavity. Based on these observations, we investigate the hypothesis that the expulsion of thermodynamically unfavorable water molecules into the bulk plays an important role in controlling the accessibility of the closed macrostate at room temperature. We compare the results of the molecular mechanics analytical generalized Born plus nonpolar solvation approach to those obtained through grid inhomogeneous solvation theory analysis with explicit solvation to elucidate the thermodynamic forces at play. The work illustrates the use of continuum solvent models to tease out solvation effects related to the inhomogeneity and the molecular nature of water and demonstrates the key role of the thermodynamics of enclosed hydration in driving the conformational equilibrium of molecules in solution.

Aggregation of amyloid-β (Aβ) peptides is a crucial step in the progression of Alzheimer's disease (AD). Identifying aggregation inhibitors against AD has been a great challenge. We report an atomistic simulation study of the inhibition mechanism of two small molecules, homotaurine and scyllo -inositol, which are AD drug candidates currently under investigation. We show that both small molecules promote a conformational change of the Aβ42 monomer toward a more collapsed phase through a nonspecific binding mechanism. This finding provides atomistic-level insights into designing potential drug candidates for future AD treatments.

Polyphenols have been suggested as potential therapeutic agents for the treatment of amyloidogenic diseases. In this work, we evaluate quercetin-rich onion extract for its ability to inhibit tau fibrillization. Considering the presence of polyphenols in multiple glycosidic and aglycosidic forms, a nanobiocatalyst-mediated approach has been used to extract quercetin from onion skins. The nanobiocatalysts facilitate greater release of quercetin compared to the use of free enzymes. Atomic force microscopy and fluorescence microscopy show that quercetin possesses a novel inhibitory character on tau-fibril aggregation. In contrast, quercetin-diglucoside does not have an inhibitory effect. Molecular Dynamics simulations reveal conformational changes in tau protein upon interaction with quercetin due to specific hydrogen bonding and hydrophobic interactions. The resulting conformational stability of tau monomer reduces propensity of the protein to aggregate. The ability of quercetin to inhibit tau fibrillization expands the paradigm for application of bioactive polyphenols., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Complexity in understanding allosteric stimulation of the hemagglutinin-neuraminidase (HN) protein of paramyxoviruses by host sialic acids (SIAs) stems from (1) unavailability of structure in its SIA-bound state and (2) the observation that this process is temperature sensitive. To consider simultaneously SIA's effect on structure and thermal fluctuations, we use molecular dynamics and simulate the dimeric form of the Newcastle disease virus HN. We find that SIA induces only minor structural changes in individual monomers, yet it reorients dimer interface by 10°. Interface reorientation is accompanied by constriction of SIA binding groove and enhanced fluctuations of interfacial residues that disrupt hydrophobic interactions and favor creation of new salt bridges. Supervised machine learning analysis of non-equilibrium data reveals that the allosteric signal is not formed from a directed sequence of these events. Altogether, we propose a detailed model of the initial events of allosteric stimulation, consistent with experiments on engineered mutations., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Signaling proteins exemplified by calmodulin usually bind cooperatively to multiple ligands. Intermediate states and allosteric behavior are difficult to characterize. Here we extend a recently reported mass spectrometry (MS)-based method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) that characterizes complex binding systems typically found as signaling proteins. As reported previously, calmodulin's response to binding four Ca 2+ can be determined by LITPOMS to reveal binding sites, binding order, and most importantly composite binding behavior. Modeling this behavior provides site-specific binding affinities. In this article, we dissect the composite, peptide-level conformational changes at several regions either by digestion with a different protease or by tandem MS of LITPOMS behavior at the amino-acid residue level. Such dissection greatly elevates spatial resolution and increases the confidence of binding-order assignment. These complementary views of complex protein conformational change recapitulate the cumulative understanding via a single approach, providing new insights on poorly understood yet important allostery and underpin an approach applicable for exploring other signaling systems.

Herein, we explored the interaction of Al 2 O 3 NPs with RBCs and Hb to determine the effect of Al 2 O 3 NPs on hemolytic activity and Hb denaturation. The percentage of hemolysis of extracts and direct contact assays triggered by Al 2 O 3 NPs was calculated by determining supernatant Hb concentration at 540 nm. Far-UV CD and Trp/ANS/acrylamide fluorescence spectroscopic methods were used to determine the structural changes of Hb upon interaction with Al 2 O 3 NPs. Theoretical studies were carried out to display the residues involved in the binding site of Hb with Al 2 O 3 nanocluster as well as the structural changes of Hb after interaction. The results showed that the percentage of hemolysis of extract and direct contact assays induced by Al 2 O 3 NPs were 1.16 and 0.46, respectively. Fluorescence spectroscopy revealed that Al 2 O 3 NPs alter the quaternary structure of the protein; however, CD spectroscopy indicated that the secondary structure of Hb remains almost unchanged. Theoretical study displayed that Al 2 O 3 nanocluster interacts with different residues of protein, and Hb tends to be destabilized at the binding site with nanocluster. This study may be significant in exploring the toxicity profile of Al 2 O 3 NPs for their in vivo implementations., (Copyright © 2019. Published by Elsevier B.V.)

Lysine-specific demethylase 1 (LSD1) has been reported to connect with a range of solid tumors. Thus, the exploration of LSD1 inhibitors has emerged as an effective strategy for cancer treatment. In this study, we constructed a pharmacophore model based on a series of flavin adenine dinucleotide (FAD)-competing inhibitors bearing triazole - dithiocarbamate scaffold combining docking, structure-activity relationship (SAR) study, and molecular dynamic (MD) simulation. Meanwhile, another pharmacophore model was also constructed manually, relying on several speculated substrate-competing inhibitors and reported putative vital interactions with LSD1. On the basis of the two pharmacophore models, multi-step virtual screenings (VSs) were performed against substrate-binding pocket and FAD-binding pocket, respectively, combining pharmacophore-based and structure-based strategy to exploit novel LSD1 inhibitors. After bioassay evaluation, four compounds among 21 hits with diverse and novel scaffolds exhibited inhibition activity at the range of 3.63-101.43 μM. Furthermore, substructure-based enrichment was performed, and four compounds with a more potent activity were identified. After that, the time-dependent assay proved that the most potent compound with IC 50 2.21 μM inhibits LSD1 activity in a manner of time-independent. In addition, the compound exhibited a cellular inhibitory effect against LSD1 in MGC-803 cells and may inhibit cell migration and invasion by reversing EMT in cultured gastric cancer cells. Considering the binding mode and SAR of the series of compounds, we could roughly deem that these compounds containing 3-methylxanthine scaffold act through occupying substrate-binding pocket competitively. This study presented a new starting point to develop novel LSD1 inhibitors.

G protein-coupled receptors (GPCRs) are highly flexible and prone to denaturation during protein extraction in detergents and purification. This poses a huge challenge to purify a conformationally homogeneous solution of GPCRs. Thermostabilizing mutations have been used widely to purify and obtain crystal structures of several GPCRs. However, identifying thermostabilizing mutations for GPCRs remains a tedious and expensive task as they are not transferable even among closely related GPCRs. Additionally, the mutations stabilizing one conformational state of a GPCR do not always stabilize other conformational state(s) of the same GPCR. Previously we developed a computational method, LiticonDesign, for rapid prediction of thermostabilizing mutations for a specific GPCR conformation. In this study, we have used LiticonDesign to predict thermostabilizing mutations for the agonist bound active-intermediate state of the human adenosine receptor (A 2A R) using the structure of the inactive state of the same GPCR and vice versa. Our study shows that the thermostable mutation predictions using LiticonDesign, for an active-intermediate state of a GPCR (A 2A R in our case), requires a homology model that is derived from an active/active-intermediate state GPCR structure as a template. Similarly, the homology models derived from inactive state GPCR conformations are better in predicting the thermostable mutations for the inactive state of A 2A R. Overall, LiticonDesign method is not only efficient in predicting thermostabilizing mutations for a given GPCR sequence but also can recover conformation specific mutations for a state of interest, if a suitable starting structure of desired conformation is chosen.

Parkinson's disease (PD), closely associated with the misfolding and aggregation of the neuronal protein α-synuclein (A-Syn), is a neurodegenerative disorder with no cure to date. Here, we show that the commercially available, inexpensive, aminoglycoside antibiotic kanamycin effectively inhibits both lipid-induced and solution-phase aggregation of A-Syn in vitro, pointing towards the prospective repurposing of kanamycin as a potential anti-PD drug.

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in the huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminal part of huntingtin (Httex1). Expanded polyQ, through a complex aggregation pathway, forms aggregates in neurons and presents a potential therapeutic target. Here we show Httex1 aggregation suppression by arginine and arginine ethyl ester (AEE) in vitro , as well as in yeast and mammalian cell models of HD, bearing expanded polyQ. These molecules also rescue locomotion dysfunction in HD Drosophila model. Both molecules alter the hydrogen bonding network of polyQ to enhance its aqueous solubility and delay aggregation. AEE shows direct binding with the NT 17 part of Httex1 to induce structural changes to impart an enhanced inhibitory effect. This study provides a platform for the development of better arginine based therapeutic molecules against polyQ-rich Httex1 aggregation.

The lantibiotic nisin is used as a food additive to effectively inactivate a broad spectrum of Gram-positive bacteria such as Listeria monocytogenes. In total, 282 L. monocytogenes field isolates from German ready-to-eat food products, food-processing environments and patient samples and 39 Listeria reference strains were evaluated for their susceptibility to nisin. The MIC 90 value was <1500 IU ml -1 . Whole genome sequences (WGS) of four nisin susceptible (NS; growth <200 IU ml -1 ) and two nisin resistant L. monocytogenes field isolates (NR; growth >1500 IU ml -1 ) of serotype IIa were analyzed for DNA sequence variants (DSVs) in genes putatively associated with NR and its regulation. WGS of NR differed from NS in the gadD2 gene encoding for the glutamate decarboxylase system (GAD). Moreover, homology modeling predicted a protein structure of GadD2 in NR that promoted a less pH dependent GAD activity and may therefore be beneficial for nisin resistance. Likewise NR had a significant faster growth rate compared to NS in presence of nisin at pH 7. In conclusion, results contributed to ongoing debate that a genetic shift in GAD supports NR state., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Caliciviruses are single-stranded RNA viruses with 180 copies of capsid protein comprising the T=3 icosahedral capsids. The main capsid feature is a pronounced protruding (P) domain dimer formed by adjacent subunits on the icosahedral surface while the shell domain forms a tight icosahedral sphere around the genome. While the P domain in the crystal structure of human Norwalk virus (genotype I.1) was tightly associated with the shell surface, the cryo-electron microscopy (cryo-EM) structures of several members of the Caliciviridae family (mouse norovirus [MNV], rabbit hemorrhagic disease virus, and human norovirus genotype II.10) revealed a "floating" P domain that hovers above the shell by nearly 10 to 15 Å in physiological buffers. Since this unusual feature is shared among, and unique to, the Caliciviridae , it suggests an important biological role. Recently, we demonstrated that bile salts enhance cell attachment to the target cell and increase the intrinsic affinity between the P domain and receptor. Presented here are the cryo-EM structures of MNV-1 in the presence of bile salts (∼3 Å) and the receptor CD300lf (∼8 Å). Surprisingly, bile salts cause the rotation and contraction of the P domain onto the shell surface. This both stabilizes the P domain and appears to allow for a higher degree of saturation of receptor onto the virus. Together, these results suggest that, as the virus moves into the gut and the associated high concentrations of bile, the entire capsid face undergoes a conformational change to optimize receptor avidity while the P domain itself undergoes smaller conformational changes to improve receptor affinity. IMPORTANCE Mouse norovirus and several other members of the Caliciviridae have been shown to have a highly unusual structure with the receptor binding protruding (P) domain only loosely tethered to the main capsid shell. Recent studies demonstrated that bile salts enhance the intrinsic P domain/receptor affinity and is necessary for cell attachment. Presented here are the high-resolution cryo-EM structures of apo MNV, MNV/bile salt, and MNV/bile salt/receptor. Bile salts cause a 90° rotation and collapse of the P domain onto the shell surface that may increase the number of available receptor binding sites. Therefore, bile salts appear to be having several effects on MNV. Bile salts shift the structural equilibrium of the P domain toward a form that binds the receptor and away from one that binds antibody. They may also cause the entire P domain to optimize receptor binding while burying a number of potential epitopes., (Copyright © 2019 American Society for Microbiology.)

Human plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids from antiatherogenic high-density lipoproteins (HDLs) to proatherogenic low-density lipoproteins (LDLs). Recent cryo-electron microscopy studies have suggested that CETP penetrates its N- and C-terminal domains in HDL and LDL to form a ternary complex, which facilitates the lipid transfer between different lipoproteins. Inhibition of CETP lipid transfer activity has been shown to increase the plasma HDL-C levels and, therefore, became an effective strategy for combating cardiovascular diseases. Thus, understanding the molecular mechanism of inhibition of lipid transfer through CETP is of paramount importance. Recently reported inhibitors, torcetrapib and anacetrapib, exhibited low potency in addition to severe side effects, which essentially demanded a thorough knowledge of the inhibition mechanism. Here, we employ steered molecular dynamics simulations to understand how inhibitors interfere with the neutral lipid transfer mechanism of CETP. Our study revealed that inhibitors physically occlude the tunnel posing a high energy barrier for lipid transfer. In addition, inhibitors bring about the conformational changes in CETP that hamper CE passage and expose protein residues that disrupt the optimal hydrophobicity of the CE transfer path. The atomic level details presented here could accelerate the designing of safe and efficacious CETP inhibitors.

The glycolytic enzyme enolase of Staphylococcus aureus is a highly conserved enzyme which binds to human plasminogen thereby aiding the infection process. The cloning, over expression and purification of S. aureus enolase as well as the effect of various metals upon the catalytic activity and structural stability of the enzyme have been reported. The recombinant enzyme (rSaeno) has been purified to homogeneity in abundant amounts (60 mg/L of culture) and the kinetic parameters (K m = 0.23 +/- 0.013 x 10 -3 M; V max = 90.98 +/- 0.00052 U/mg) and the optimum pH were calculated. This communication further reports that increasing concentrations of Na + ions inhibit the enzyme while increasing concentrations of K + ions were stimulatory. In case of divalent cations, it was found that Mg 2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn 2+ , Mn 2+ , Fe 2+ , Cu 2+ , Ni 2+ and Ca 2+ ) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg 2+ and Cr 2+ . The circular dichroism data indicate that other than Hg 2+ , Ni 2+ and to a certain extent Cu 2+ , none of the other ions destabilized rSaeno. The inhibitory roles of fluorides, as well as neurotoxic compounds upon the catalytic activity of rSaeno, have also been studied. Conformational changes in rSaeno (induced by ions) were studied using partial trypsin digestion.

Natalizumab (NZM), a humanized monoclonal IgG4 antibody to α4 integrins, is used to treat patients with relapsing-remitting multiple sclerosis (MS) 1,2 , but in about 6% of the cases persistent neutralizing anti-drug antibodies (ADAs) are induced leading to therapy discontinuation 3,4 . To understand the basis of the ADA response and the mechanism of ADA-mediated neutralization, we performed an in-depth analysis of the B and T cell responses in two patients. By characterizing a large panel of NZM-specific monoclonal antibodies, we found that, in both patients, the response was polyclonal and targeted different epitopes of the NZM idiotype. The neutralizing activity was acquired through somatic mutations and correlated with a slow dissociation rate, a finding that was supported by structural data. Interestingly, in both patients, the analysis of the CD4 + T cell response, combined with mass spectrometry-based peptidomics, revealed a single immunodominant T cell epitope spanning the FR2-CDR2 region of the NZM light chain. Moreover, a CDR2-modified version of NZM was not recognized by T cells, while retaining binding to α4 integrins. Collectively, our integrated analysis identifies the basis of T-B collaboration that leads to ADA-mediated therapeutic resistance and delineates an approach to design novel deimmunized antibodies for autoimmune disease and cancer treatment.

Snake venom cardiotoxins (CTXs) present diverse pharmacological functions. Previous studies have reported that CTXs affect the activity of some serine proteases, namely, chymotrypsin, subtilisin, trypsin, and acetylcholinesterase. To elucidate the mode of action of CTXs, the interaction of CTXs with chymotrypsin was thus investigated. It was found that Naja atra CTX isotoxins concentration-dependently enhanced chymotrypsin activity. The capability of CTX1 and CTX5 in increasing chymotrypsin activity was higher than that of CTX2, CTX3, and CTX4. Removal of the molecular beacon-bound CTXs by chymotrypsin, circular dichroism measurement, and acrylamide quenching of Trp fluorescence indicated that CTXs bound to chymotrypsin. Chemical modification of Lys, Arg, or Met residues of CTX1 attenuated its capability to enhance chymotrypsin activity without impairing their bond with chymotrypsin. Catalytically inactive chymotrypsin retained the binding affinity for native and modified CTX1. CTX1 and chemically modified CTX1 differently altered the global conformation of chymotrypsin and inactivated chymotrypsin. Moreover, CTX1 did not reduce the interaction of 2-(p-toluidino)-naphthalene-6-sulfonate (TNS) with chymotrypsin and inactivated chymotrypsin. Together with previous results revealing that TNS can bind at the hydrophobic region of active site in chymotrypsin, our data suggest that CTXs can enhance chymotrypsin activity by binding to the region outside the enzyme's active site., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A number of viruses causing sexually transmissible diseases are transmitted via mammalian seminal plasma. Several components of seminal plasma have been shown to influence those viruses and their physiological impact. To unravel whether components of seminal plasma could affect viruses transmitted via other pathways, it was investigated here whether the bovine seminal plasma protein PDC-109, belonging to the Fn-type 2 protein family, influences the activity of influenza A viruses, used as a model for enveloped viruses. We found that PDC-109 inhibits the fusion of influenza virus with human erythrocyte membranes and leads to a decreased viral infection in MDCK cells. In the presence of the head group of the phospholipid phosphatidylcholine, phosphorylcholine, the inhibitory effect of PDC-109 was attenuated. This indicates that the impact of the protein is mainly caused by its binding to viral and to erythrocyte membranes thereby interfering with virus-cell binding. Our study underlines that Fn-type 2 proteins have to be considered as new antiviral components present in mammalian seminal plasma.

Previous studies in our laboratory characterized a panel of highly mutated HIV-specific conformational epitope-targeting antibodies (Abs) from a panel of HIV-infected long-term nonprogressors (LTNPs). Despite binding HIV envelope protein and having a high number of somatic amino acid mutations, these Abs had poor neutralizing activity. Because of the evidence of antigen-driven selection and the long CDR3 region (21 amino acids [aa]), we further characterized the epitope targeting of monoclonal Ab (MAb) 76-Q3-2C6 (2C6). We confirmed that 2C6 binds preferentially to trimeric envelope and recognizes the clades A, B, and C SOSIP trimers. 2C6 binds gp140 constructs of clades A, B, C, and D, suggesting a conserved binding site that we localized to the ectodomain of gp41. Ab competition with MAb 50-69 suggested this epitope localizes near aa 579 to 613 (referenced to HXB2 gp160). Peptide library scanning showed consistent binding in this region but to only a single peptide. Lack of overlapping peptide binding supported a nonlinear epitope structure. The significance of this site is supported by 2C6 having Ab-dependent cell cytotoxicity (ADCC) against envelope proteins from two clades. Using 2C6 and variants, alanine scanning mutagenesis identified three amino acids (aa 592, 595, and 596) in the overlapping region of the previously identified peptide. Additional amino acids at sites 524 and 579 were also identified, helping explain its conformational requirement. The fact that different amino acids were included in the epitope depending on the targeted protein supports the conclusion that 2C6 targets a native conformational epitope. When we mapped these amino acids on the trimerized structure, they spanned across oligomers, supporting the notion that the epitope targeted by 2C6 lies in a recessed pocket between two gp41 oligomers. A complete understanding of the epitope specificity of ADCC-mediating Abs is essential for developing effective immunization strategies that optimize protection by these Abs. IMPORTANCE This paper further defines the function and area of the HIV trimeric envelope protein targeted by the monoclonal antibody 2C6. 2C6 binding is influenced by amino acid mutations across two separate gp41 sections of the envelope trimer. This epitope is recognized on multiple clades (variant groups of circulating viruses) of gp41, gp140 trimers, and SOSIP trimers. For the clades tested, 2C6 has robust ADCC. As the target of 2C6 is available in the major clades of HIV and has robust ADCC activity, further definition and appreciation of targeting of antibodies similar to 2C6 during vaccine development should be considered., (Copyright © 2019 American Society for Microbiology.)

Enveloped viruses utilize surface glycoproteins to bind and fuse with a target cell membrane. The zoonotic Hendra virus (HeV), a member of the family Paramyxoviridae , utilizes the attachment protein (G) and the fusion protein (F) to perform these critical functions. Upon triggering, the trimeric F protein undergoes a large, irreversible conformation change to drive membrane fusion. Previously, we have shown that the transmembrane (TM) domain of the F protein, separate from the rest of the protein, is present in a monomer-trimer equilibrium. This TM-TM association contributes to the stability of the prefusion form of the protein, supporting a role for TM-TM interactions in the control of F protein conformational changes. To determine the impact of disrupting TM-TM interactions, constructs expressing the HeV F TM with limited flanking sequences were synthesized. Coexpression of these constructs with HeV F resulted in dramatic reductions in the stability of F protein expression and fusion activity. In contrast, no effects were observed when the HeV F TM constructs were coexpressed with the nonhomologous parainfluenza virus 5 (PIV5) fusion protein, indicating a requirement for specific interactions. To further examine this, a TM peptide homologous to the PIV5 F TM domain was synthesized. Addition of the peptide prior to infection inhibited infection with PIV5 but did not significantly affect infection with human metapneumovirus, a related virus. These results indicate that targeted disruption of TM-TM interactions significantly impact viral fusion protein stability and function, presenting these interactions as a novel target for antiviral development. IMPORTANCE Enveloped viruses require virus-cell membrane fusion to release the viral genome and replicate. The viral fusion protein triggers from the pre- to the postfusion conformation, an essentially irreversible change, to drive membrane fusion. We found that small proteins containing the TM and a limited flanking region homologous to the fusion protein of the zoonotic Hendra virus reduced protein expression and fusion activity. The introduction of exogenous TM peptides may displace a TM domain, disrupting native TM-TM interactions and globally destabilizing the fusion protein. Supporting this hypothesis, we showed that a sequence-specific transmembrane peptide dramatically reduced viral infection in another enveloped virus model, suggesting a broader inhibitory mechanism. Viral fusion protein TM-TM interactions are important for protein function, and disruption of these interactions dramatically reduces protein stability., (Copyright © 2019 American Society for Microbiology.)

Ertugliflozin is a potent and selective inhibitor of sodium-dependent glucose cotransporters 2 (SGLT2) and used as a monotherapy to improve glycemic control in adult patients with type 2 diabetes. In this study, ertugliflozin binding to human serum albumin (HSA) was investigated by multispectroscopic and computer simulations. The fluorescence spectra demonstrated that the quenching mechanism of ertugliflozin and HSA was static quenching. Thermodynamic parameters indicated that hydrogen bonding and van der Waals forces played a key role in the binding. Fluorescence competition experiments and molecular docking revealed that ertugliflozin bound to HSA sites II. In three-dimensional fluorescence, circular dichroism spectroscopy, and molecular dynamics simulation, ertugliflozin did not affect the basic skeleton structure of HSA but slightly increased the α-helical structure content and changed the microenvironment around amino acid residues. Results provide valuable information on the basis of the interaction of ertugliflozin with HSA., (Copyright © 2019. Published by Elsevier B.V.)

The interactions between cetyltrimethylammonium bromide (CTAB) and hen egg white lysozymes (HEWL) was carried out to investigate protein-surfactant interaction mechanisms while both exist in the overall same charged state. The interactions between CTAB and the HEWL were examined with circular dichroism (CD), dynamic light scattering (DLS), fluorescence spectroscopy, and computational docking at a pH9.0 at room temperature. The far-UV CD and fluorescence results revealed that CTAB at concentrations from 0.15 to 10.0mM influenced the secondary as well as the tertiary structure of HEWL. The secondary structure of the HEWL was retained, while the tertiary structure of the HEWL was disrupted in the CTAB-treated samples at pH9.0. The hydrodynamic radii of the HEWL were also expanded in the presence of CTAB. Molecular docking studies showed that CTAB formed one electrostatic and four hydrophobic interactions, as well as one carbon hydrogen bond with HEWL. The data obtained from spectroscopic and computational studies demonstrated that the positively charged head and 18‑carbon alkyl chain of the CTAB interacted through weak electrostatic and strong hydrophobic interactions., (Copyright © 2019. Published by Elsevier B.V.)

High glucose can lead to toxicity on islet β cells. The protective effects of a novel Lentinus edodes mycelia polysaccharide (LMP) on INS-1 cells damaged by glucose were investigated. Cell viability, lactate dehydrogenase (LDH) release, cell apoptosis, intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were detected. P38 MAPK, JNKand NF-κB pathways were analyzed to reveal the inhibitory mechanism of LMP on glucose-induced INS-1 cells toxicity. The results showed that LMP could decrease cellular oxidative stress, reduce intracellular ROS levels, decrease MDA content and increase SOD activity. Furthermore, the glucose-induced cell apoptosis in cells were inhibited by regulating the expression of Bax, Bcl-2, cleaved caspase‑3 and cleaved caspase‑1. Cell signaling pathway analysis revealed that LMP could inhibit the activation of p38 MAPK, JNK, NF-κB pathways and activate Nrf2 pathway. To further explore the possible transportation mechanism of LMP with human serum albumin (HSA), ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy were used to evaluate the interaction between LMP and HSA. The results showed that LMP-HSA complex was formed, which would be helpful for explaining the transportation mechanism in vivo. These results suggested that LMP might be a new therapeutic candidate to alleviate the high glucose toxicity., (Copyright © 2019. Published by Elsevier B.V.)

Heavy metal(loid)s such as Cd and Hg adversely affect human health and are therefore strictly regulated and monitored; however, their quantitation in the environment is usually performed by expensive and time-consuming instrumental analysis techniques, which necessitates the search for more practical alternatives. Herein, we prepare enhanced green fluorescent protein (eGFP)-based biomolecules for metal(loid) sensing by insertion of metal-binding loops (MBLs) into a loop region of eGFP to render this protein inactive and show that the binding of metal ions to MBLs induces a conformational change and restores the original activity. Specifically, eGFP with an MBL sequenced as CTTCGCG regains fluorescence upon exposure to Cd and Hg, which allows the above metals to be quantified in the concentration range of 0-5 μM. For practical applicability verification, the developed sensing platform is used to quantify Cd in artificially amended soil and water samples. Although the obtained results imply that sensor performance needs to be significantly improved, the presented design concept is believed to be of high value to researchers in the field of heavy metal sensing and facilitate the development of new biosensors.

ABCA3 is a phospholipid transporter implicated in pulmonary surfactant homoeostasis and localized at the limiting membrane of lamellar bodies, the storage compartment for surfactant in alveolar type II cells. Mutations in ABCA3 display a common genetic cause for diseases caused by surfactant deficiency like respiratory distress in neonates and interstitial lung disease in children and adults, for which currently no causal therapy exists. In this study, we investigated the effects of ivacaftor and genistein, two potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR), on ABCA3-specific lipid transport function. Wild-type (WT) and functional ABCA3 mutations N568D, F629L, G667R, T1114M and L1580P were stably expressed in A549 cells. Three-dimensional modelling predicted functional impairment for all five mutants that was confirmed by in vitro experiments (all <14% of WT functional activity). Treatment with potentiators rescued the mutants N568D (up to 114% of WT), F629L (up to 47% of WT), and G667R (up to 60% of WT), the latter variation needing higher concentrations of genistein, showing reduced affinity of the potentiator to the mutant protein. Our results present a first proof that functional ABCA3 mutations are rescued by CFTR potentiators, making them a potential therapeutical option for patients suffering from surfactant deficiency due to ABCA3 mutations., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Staphylococcus aureus is an important cause of resistant healthcare-associated infections. It has been shown that the wall teichoic acid (WTA) may be an important drug target acting on antibiotic-resistant cells. The UDP-N-acetylglucosamine 2-epimerase, MnaA, is one of the first enzymes on the pathway for the biosynthesis of the WTA. Here, detailed molecular dynamics simulations of S. aureus MnaA were used to characterize the conformational changes that occur in the presence of UDP and UDP-GlcNac and also the energetic landscape associated with these changes. Using different simulation techniques, such as ABMD and GAMD, it was possible to assess the energetic profile for the protein with and without ligands in its active site. We found that there is a dynamic energy landscape that has its minimum changed by the presence of the ligands, with a closed structure of the enzyme being more frequently observed for the bound state while the unbound enzyme favors an opened conformation. Further structural analysis indicated that positively charged amino acids associated with UDP and UDP-GlcNac interactions play a major role in the enzyme opening movement. Finally, the energy landscape profiled in this work provides important conclusions for the design of inhibitor candidates targeting S. aureus MnaA., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The 2.5 Å structure of the cytochrome (cyt) b 6 f complex provides a basis for control of the rate-limiting electron transfer step of oxygenic photosynthesis associated with the plastoquinol/quinone exchange pathway. Here, a structural change was made at a site containing two proline residues which border the intra-cyt pathway for plastoquinol/quinone exchange. The proline side chains confer a larger aperture for passage of plastoquinol/quinone. Change of these prolines to alanine in the cyanobacterium Synechococcus sp. PCC 7002 results in attenuation of this rate-limiting step, observed by a two-fold reduction in the rate of cell growth, O 2 evolution, and plastoquinol-mediated reduction of cyt f. This study demonstrates modification by site-directed mutagenesis of photosynthetic energy transduction based on rational application of information in the atomic structure., (© 2019 Federation of European Biochemical Societies.)

The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is the best recognized and most widely implicated regulator of caspase-1 activation. It is a key regulator of innate immune response and is involved in many pathophysiological processes. Recent evidences for its inappropriate activation in autoinflammatory, autoimmune, as well as in neurodegenerative diseases attract a growing interest toward the development of small molecules NLRP3 inhibitors. Based on the knowledge of biochemical and structural aspects of NLRP3 activation, one successful strategy in the identification of NLRP3 inhibitors relies on the development of covalent irreversible inhibitors. Covalent inhibitors are reactive electrophilic molecules able to alkylate nucleophiles in the target protein. These inhibitors could ensure good efficacy and prolonged duration of action both in vitro and in vivo. In spite of these advantages, effects on other signalling pathways, prone to alkylation, may occur. In this review, we will illustrate the chemistry and the biological action of the most studied covalent NLRP3 inhibitors developed so far. A description of what we know about their mechanism of action will address the reader toward a critical understanding of NLRP3 inhibition by electrophilic compounds., (Copyright © 2018 Elsevier Inc. All rights reserved.)