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T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing β-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity.

P2X receptors are a class of nonselective cation channels widely distributed in the immune and nervous systems, and their dysfunction is a significant cause of tumors, inflammation, leukemia, and immune diseases. P2X7 is a unique member of the P2X receptor family with many properties that differ from other subtypes in terms of primary sequence, the architecture of N- and C-terminals, and channel function. Here, we suggest that the observed lengthened β2- and β3-sheets and their linker (loop β2,3), encoded by redundant sequences, play an indispensable role in the activation of the P2X7 receptor. We show that deletion of this longer structural element leads to the loss of P2X7 function. Furthermore, by combining mutagenesis, chimera construction, surface expression, and protein stability analysis, we found that the deletion of the longer β2,3-loop affects P2X7 surface expression but, more importantly, that this loop affects channel gating of P2X7. We propose that the longer β2,3-sheets may have a negative regulatory effect on a loop on the head domain and on the structural element formed by E171 and its surrounding regions. Understanding the role of the unique structure of the P2X7 receptor in the gating process will aid in the development of selective drugs targeting this subtype., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

For an efficacious vaccine immunogen, influenza hemagglutinin (HA) needs to maintain a stable quaternary structure, which is contrary to the inherently dynamic and metastable nature of class I fusion proteins. In this study, we stabilized HA with three substitutions within its pH-sensitive regions where the refolding starts. An X-ray structure reveals how these substitutions stabilize the intersubunit β-sheet in the base and form an interprotomeric aliphatic layer across the stem while the native prefusion HA fold is retained. The identification of the stabilizing substitutions increases our understanding of how the pH sensitivity is structurally accomplished in HA and possibly other pH-sensitive class I fusion proteins. Our stabilization approach in combination with the occasional back mutation of rare amino acids to consensus results in well-expressing stable trimeric HAs. This repair and stabilization approach, which proves broadly applicable to all tested influenza A HAs of group 1 and 2, will improve the developability of influenza vaccines based on different types of platforms and formats and can potentially improve efficacy., Competing Interests: Competing interest statement: F.J.M., M.J., T.R., B.B., and J.P.M.L. are coinventors on related vaccine patents. F.J.M., M.J., T.R., P.B., I.J.M.B., M.d.M., D.V., L.L., B.K., M.J.G.B., J.J., B.B., and J.P.M.L. are employees of Janssen Vaccines & Prevention. M.J., J.J., B.B., P.B., and J.P.M.L. hold stock of Johnson & Johnson., (Copyright © 2022 the Author(s). Published by PNAS.)

TAR DNA-binding protein-43 (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). Hyperphosphorylation and aggregation of TDP-43 contribute to pathology and are viable therapeutic targets for ALS. In vivo inhibition of TDP-43 aggregation was evaluated using anti-TDP-43 antibodies with promising outcomes. However, the exact mechanism of antibody-based inhibition targeting TDP-43 is not well understood but may lead to the identification of viable immunotherapies. Herein, the mechanism of in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Specifically, the aggregation of phosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) spectroscopy. The protein aggregates were insoluble, ThT-positive and characterized with heterogeneous morphologies (fibers, amorphous structures). Antibodies specific to epitopes 178-393 and 256-269, within the RRM2-CTD domain, reduced the formation of β-sheets and insoluble aggregates, at low antibody loading (antibody: protein ratio = 1 μg/mL: 45 μg/mL). Inhibition outcomes were highly dependent on the type and loading of antibodies, indicating dual functionality of such inhibitors, as aggregation inhibitors or aggregation promoters. Anti-SOD1 and anti-tau antibodies were not effective inhibitors against TDP-43 aggregation, indicating selective inhibition., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is the second most common neurodegenerative disorder. An important hallmark of PD involves the pathological aggregation of proteins in structures known as Lewy bodies. The major component of these proteinaceous inclusions is alpha (α)-synuclein. In different conditions, α-synuclein can assume conformations rich in either α-helix or β-sheets. The mechanisms of α-synuclein misfolding, aggregation, and fibrillation remain unknown, but it is thought that β-sheet conformation of α-synuclein is responsible for its associated toxic mechanisms. To gain fundamental insights into the process of α-synuclein misfolding and aggregation, the secondary structure of this protein in the presence of charged and non-charged surfactant solutions was characterized. The selected surfactants were (anionic) sodium dodecyl sulphate (SDS), (cationic) cetyltrimethylammonium chloride (CTAC), and (uncharged) octyl β-D-glucopyranoside (OG). The effect of surfactants in α-synuclein misfolding was assessed by ultra-structural analyses, in vitro aggregation assays, and secondary structure analyses. The α-synuclein aggregation in the presence of negatively charged SDS suggests that SDS-monomer complexes stimulate the aggregation process. A reduction in the electrostatic repulsion between N- and C-terminal and in the hydrophobic interactions between the NAC (non-amyloid beta component) region and the C-terminal seems to be important to undergo aggregation. Fourier transform infrared spectroscopy (FTIR) measurements show that β-sheet structures comprise the assembly of the fibrils.

Rapid and sensitive detection of Salmonella is a critical step in routine food quality control, outbreak investigation, and food recalls. Although various genes have been the targets in the design of rapid molecular detection methods for Salmonella, there is limited information on the diversity of these target genes at the level of DNA sequence and the encoded protein structures. In this study, we investigated the diversity of ten target genes (invA, fimA, phoP, spvC, and agfA; ttrRSBCA operon including 5 genes) commonly used in the detection and identification of Salmonella. To this end, we performed whole genome sequencing of 143 isolates of Salmonella serotypes (Enteritidis, Typhimurium, and Heidelberg) obtained from poultry (eggs and chicken). Phylogenetic analysis showed that Salmonella ser. Typhimurium was more diverse than either Enteritidis or Heidelberg. Forty-five non-synonymous mutations were identified in the target genes from the 143 isolates, with the two most common mutations as T ↔ C (15 times) and A ↔ G (13 times). The gene spvC was primarily present in Salmonella ser. Enteritidis isolates and absent from Heidelberg isolates, whereas ttrR was more conserved (0 non-synonymous mutations) than ttrS, ttrB, ttrC, and ttrA (7, 2, 2, and 7 non-synonymous mutations, respectively). Notably, we found one non-synonymous mutation (fimA-Mut.6) across all Salmonella ser. Enteritidis and Salmonella ser. Heidelberg, C → T (496 nt postion), resulting in the change at AA 166 position, Glutamine (Q) → Stop condon (TAG), suggesting that the fimA gene has questionable sites as a target for detection. Using Phyre 2 and SWISS-MODEL software, we predicted the structures of the proteins encoded by some of the target genes, illustrating the positions of these non-synonymous mutations that mainly located on the α-helix and β-sheet which are key elements for maintaining the conformation of proteins. These results will facilitate the development of sensitive molecular detection methods for Salmonella., (© 2021. The Author(s).)

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for most mitochondrial proteins. The TOM complex is a multisubunit membrane protein complex consisting of a β-barrel protein Tom40 and six α-helical transmembrane (TM) proteins, receptor subunits Tom20, Tom22, and Tom70, and regulatory subunits Tom5, Tom6, and Tom7. Although nearly 30 years have passed since the main components of the TOM complex were identified and characterized, the structural details of the TOM complex remained poorly understood until recently. Thanks to the rapid development of the cryoelectron microscopy (EM) technology, high-resolution structures of the yeast TOM complex have become available. The identified structures showed a symmetric dimer containing five different subunits including Tom22. Biochemical and mutational analyses based on the TOM complex structure revealed the presence of different translocation paths within the Tom40 import channel for different classes of translocating precursor proteins. Previous studies including our cross-linking analyses indicated that the TOM complex in intact mitochondria is present as a mixture of the trimeric complex containing Tom22. Furthermore, the dimeric complex lacking Tom22, and the trimer and dimer may handle different sets of mitochondrial precursor proteins for translocation across the outer membrane. In this Structural Snapshot, we will discuss possible rearrangement of the subunit interactions upon dynamic conversion of the TOM complex between the different subunit assembly states, the Tom22-containing core dimer and trimer., (© 2020 Federation of European Biochemical Societies.)

The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC's ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity.

Eukaryotic cells express thousands of protein domains long believed to function in the absence of molecular order. These intrinsically disordered protein (IDP) domains are typified by gibberish-like repeats of only a limited number of amino acids that we refer to as domains of low sequence complexity. A decade ago, it was observed that these low complexity (LC) domains can undergo phase transition out of aqueous solution to form either liquid-like droplets or hydrogels. The self-associative interactions responsible for phase transition involve the formation of specific cross-β structures that are unusual in being labile to dissociation. Here we give evidence that the LC domains of two RNA binding proteins, ataxin-2 and TDP43, form cross-β interactions that specify biologically relevant redox sensors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.

Studies have established that congenital cataract is the major cause of blindness in children across the globe. The β-crystallin protein family is the richest and most soluble structural protein in the lens. Their solubility and stability are essential in maintaining lens transparency. In this study, we identified a novel βB2 mutation W151R in a rare progressive cortical congenital cataract family and explored its pathogenesis using purified protein and mutant related cataract-cell models. Due to its low solubility and poor structural stability, the βB2 W151R mutation was prone to aggregation. Moreover, the W151R mutation enhanced the exposure of the hydrophobic side chains in the fourth Greek Key motif, which were readily degraded by trypsin. However, upon the administration of lanosterol, the negative effect of the W151R mutation was reversed. Therefore, lanosterol is a potential therapeutic option for cataracts., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The amyloid-β (Aβ) peptides are associated with two prominent diseases in the brain, Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). Aβ42 is the dominant component of cored parenchymal plaques associated with AD, while Aβ40 is the predominant component of vascular amyloid associated with CAA. There are familial CAA mutations at positions Glu22 and Asp23 that lead to aggressive Aβ aggregation, drive vascular amyloid deposition and result in degradation of vascular membranes. In this study, we compared the transition of the monomeric Aβ40-WT peptide into soluble oligomers and fibrils with the corresponding transitions of the Aβ40-Dutch (E22Q), Aβ40-Iowa (D23N) and Aβ40-Dutch, Iowa (E22Q, D23N) mutants. FTIR measurements show that in a fashion similar to Aβ40-WT, the familial CAA mutants form transient intermediates with anti-parallel β-structure. This structure appears before the formation of cross-β-sheet fibrils as determined by thioflavin T fluorescence and circular dichroism spectroscopy and occurs when AFM images reveal the presence of soluble oligomers and protofibrils. Although the anti-parallel β-hairpin is a common intermediate on the pathway to Aβ fibrils for the four peptides studied, the rate of conversion to cross-β-sheet fibril structure differs for each.

We designed three types of RGD-containing barnacle adhesive proteins using self-assembling peptides. In the present study, three types of RGD-containing peptides were synthesized by solid-phase peptide synthesis, and the secondary structures of these peptides were analyzed by CD and FT-IR spectroscopy. The mechanical properties of peptide hydrogels were characterized by a rheometer. We discuss the correlation between the peptide conformation, and cell attachment and cell spreading activity from the viewpoint of developing effective tissue engineering scaffolds. We created a peptide-coated cell culture substrate by coating peptides on a polystyrene plate. They significantly facilitated cell adhesion and spreading compared to a non-coated substrate. When the RGDS sequence was modified at N- or C-terminal of R-Y, it was found that the self-assembling ability was dependent on the strongly affects hydrogel formation and cell adhesion caused by its secondary structure.

Hedgehog signaling plays a crucial role in embryogenesis and adult tissue homeostasis, and mutations of its key components such as Suppressor of fused (Sufu) are closely associated with human diseases. The Ser/Thr kinase Fused (Fu) promotes Hedgehog signaling by phosphorylating the Cubitus interruptus (Ci)/Glioma-associated oncogene homologue (Gli) family of transcription factors. Sufu associates with both Fu and Ci/Gli, but the recognition mechanism between Sufu and Fu remains obscure. Here, our structure of the N-terminal domain (NTD) of Drosophila Sufu (dSufu) in complex with the Sufu-binding site (SBS) of Fu reveals that both main-chain β sheet formation and side-chain hydrophobic interactions contribute to the recognition between Sufu and Fu, and point mutations of highly conserved interface residues eliminated their association. Structural comparison suggests that Fu and Ci/Gli bind on opposite sides of dSufu-NTD, allowing the formation of a Fu-dSufu-Ci ternary complex which facilitates the phosphorylation of Ci/Gli by Fu. Hence, our results provide insights into the Sufu-Fu recognition mechanism., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Members of the Herpesviridae, including the medically important alphaherpesvirus varicella-zoster virus (VZV), induce fusion of the virion envelope with cell membranes during entry, and between cells to form polykaryocytes in infected tissues. The conserved glycoproteins, gB, gH and gL, are the core functional proteins of the herpesvirus fusion complex. gB serves as the primary fusogen via its fusion loops, but functions for the remaining gB domains remain unexplained. As a pathway for biological discovery of domain function, our approach used structure-based analysis of the viral fusogen together with a neutralizing antibody. We report here a 2.8 Å cryogenic-electron microscopy structure of native gB recovered from VZV-infected cells, in complex with a human monoclonal antibody, 93k. This high-resolution structure guided targeted mutagenesis at the gB-93k interface, providing compelling evidence that a domain spatially distant from the gB fusion loops is critical for herpesvirus fusion, revealing a potential new target for antiviral therapies.

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.

Anti-cancer peptides (ACPs) are a series of short peptides composed of 10-60 amino acids that can inhibit tumour cell proliferation or migration, or suppress the formation of tumour blood vessels, and are less likely to cause drug resistance. The aforementioned merits make ACPs the most promising anti-cancer candidate. However, ACPs may be degraded by proteases, or result in cytotoxicity in many cases. To overcome these drawbacks, a plethora of research has focused on reconstruction or modification of ACPs to improve their anti-cancer activity, while reducing their cytotoxicity. The modification of ACPs mainly includes main chain reconstruction and side chain modification. After summarizing the classification and mechanism of action of ACPs, this paper focuses on recent development and progress about their reconstruction and modification. The information collected here may provide some ideas for further research on ACPs, in particular their modification.

GABA A receptors (GABA A Rs) mediate inhibitory neurotransmission in the mammalian brain. Recently, numerous GABA A R static structures have been published, but the molecular mechanisms of receptor activation remain elusive. Loop G is a rigid β-strand belonging to an extensive β-sheet that spans the regions involved in GABA binding and the interdomain interface which is important in receptor gating. It has been reported that loop G participates in ligand binding and gating of GABA A Rs, however, it remains unclear which specific gating transitions are controlled by this loop. Analysis of macroscopic responses revealed that mutation at the α 1 F45 residue (loop G midpoint) resulted in slower macroscopic desensitization and accelerated deactivation. Single-channel analysis revealed that these mutations also affected open and closed times distributions and reduced open probability. Kinetic modeling demonstrated that mutations affected primarily channel opening/closing and ligand binding with a minor effect on preactivation. Thus, α 1 F45 residue, in spite of its localization close to binding site, affects late gating transitions. In silico structural analysis suggested an important role of α 1 F45 residue in loop G stability and rigidity as well as in general structure of the binding site. We propose that the rigid β-sheet comprising loop G is well suited for long range communication within GABA A R but this mechanism becomes impaired when α 1 F45 is mutated. In conclusion, we demonstrate that loop G is crucial in controlling both binding and gating of GABA A Rs. These data shed new light on GABA A R activation mechanism and may also be helpful in designing clinically relevant modulators., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Glucagon is a 29 amino acid peptide hormone secreted by pancreatic α-cells that interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibrillar aggregates that are cytotoxic due to their activation of apoptotic signaling pathways. To understand the glucagon-membrane interactions, morphological changes in dimyristoylphosphatidylcholine (DMPC) bilayers containing glucagon in neutral solution were investigated by observing 31 P NMR spectra. First, lipid bilayers with a DMPC/glucagon molar ratio of 50/1 were observed. One day after preparing the DMPC/glucagon lipid bilayer sample, lipid bilayers were disrupted below the phase transition temperature (Tc). Membrane disruption was reduced 2 days after preparation due to the reduction of glucagon-DMPC interaction, and subsequently increased by 4 days and was reduced again by 7 days. TEM measurements showed that small ellipsoidal intermediates of glucagon were observed inside the small size of lipid bilayer after 4 days, while fibrils grew inside lipid bilayer after 19 days. These results indicate that morphological changes in DMPC/glucagon lipid bilayers are correlated with the evolution of glucagon aggregate state. Particularly, fibril intermediate shows a strong glucagon lipid bilayer interaction. We further investigated the structure and kinetics of glucagon fibril formation inside the DMPC lipid bilayer in a neutral solution using 13 C solid-state NMR spectroscopy. α-Helical structures were observed around Gly4 and Ala19 in the monomeric form, which changed to β-sheet structures in the fibril form. The fibrillation process can be explained by a two-step autocatalytic reaction mechanism in which the first step is a homogeneous nuclear formation (k 1 ), and the second step is an autocatalytic heterogeneous fibrillation process (k 2 )., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.

Xylanase B, a member of subfamily 7 of the GH30 (glycoside hydrolase family 30) from Talaromyces cellulolyticus (TcXyn30B), is a bifunctional enzyme with glucuronoxylanase and xylobiohydrolase activities. In the present study, crystal structures of the native enzyme and the enzyme-product complex of TcXyn30B expressed in Pichia pastoris were determined at resolutions of 1.60 and 1.65 Å, respectively. The enzyme complexed with 2 2 -(4-O-methyl-α-d-glucuronyl)-xylobiose (U 4m2 X) revealed that TcXyn30B strictly recognizes both the C-6 carboxyl group and the 4-O-methyl group of the 4-O-methyl-α-d-glucuronyl side chain by the conserved residues in GH30-7 endoxylanases. The crystal structure and site-directed mutagenesis indicated that Asn-93 on the β2-α2-loop interacts with the non-reducing end of the xylose residue at subsite-2 and is likely to be involved in xylobiohydrolase activity. These findings provide structural insight into the mechanisms of substrate recognition of GH30-7 glucuronoxylanase and xylobiohydrolase., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

The essentiality of DNA Gyrase in basic cellular processes in bacterial pathogens makes it an ideal drug target. Though the Gyrase has a conserved mechanism of action, the complete DNA wrapping and binding process is still unknown. In this study, we have identified six arginine residues R556, R612, R667, R716, R766, and R817 in the DNA GyraseA - C-terminal domain from Salmonella enterica serovar Typhi (StGyrA-CTD) to be essential for DNA wrapping and sliding by a sequence and structure analysis. Through site-directed mutagenesis and EMSA studies, we observed that the substitution of R667 (blade 3) and R716 (blade 4) in StGyrA-CTD led to loss of DNA binding. Whereas, upon mutation of residue R612 (blade2), R766 (blade5) and R817 (blade6) along with supporting residue R712 (blade 4) a decrease in binding affinity was seen. Our results indicate that R667 and R716 act as a pivot point in DNA wrapping and sliding during gyrase catalytic activity. In this study, we propose that the DNA wrapping mechanism commences with DNA binding at blade3 and blade4 followed by other blades to facilitate the DNA sliding during supercoiling activity. This study provides a better understanding of the DNA binding and wrapping mechanism of GyrA-CTD in DNA Gyrase.

Amyloids are protein aggregates observed in several diseases, for example in Alzheimer's and Parkinson's diseases. An aggregate has a very regular beta structure with a tightly packed core, which spontaneously assumes a steric zipper form. Experimental methods enable studying such peptides, however they are tedious and costly, therefore inappropriate for genomewide studies. Several bioinformatic methods have been proposed to evaluate protein propensity to form an amyloid. However, the knowledge of aggregate structures is usually not taken into account. We propose PATH (Prediction of Amyloidogenicity by THreading) - a novel structure-based method for predicting amyloidogenicity and show that involving available structures of amyloidogenic fragments enhances classification performance. Experimental aggregate structures were used in templatebased modeling to recognize the most stable representative structural class of a query peptide. Several machine learning methods were then applied on the structural models, using their energy terms. Finally, we identified the most important terms in classification of amyloidogenic peptides. The proposed method outperforms most of the currently available methods for predicting amyloidogenicity, with its area under ROC curve equal to 0.876. Furthermore, the method gave insight into significance of selected structural features and the potentially most stable structural class of a peptide fragment if subjected to crystallization.

Angiotensin-converting enzyme (ACE) is best known for its formation of the vasopressor angiotensin II that controls blood pressure but is also involved in other physiological functions through the hydrolysis of a variety of peptide substrates. The enzyme contains two catalytic domains (nACE and cACE) that have different affinities for ACE substrates and inhibitors. We investigated whether nACE inhibitor backbones contain a unique property which allows them to take advantage of the hinging of nACE. Kinetic analysis showed that mutation of unique nACE residues, in both the S2 pocket and around the prime subsites (S') to their C-domain counterparts, each resulted in a decrease in the affinity of nACE specific inhibitors (SG6, 33RE and ketoACE-13) but it required the combined S2_S' mutant to abrogate nACE-selectivity. However, this was not observed with the non-domain-selective inhibitors enalaprilat and omapatrilat. High-resolution structures were determined for the minimally glycosylated nACE with the combined S2_S' mutations in complex with the ACE inhibitors 33RE (1.8 Å), omapatrilat (1.8 Å) and SG6 (1.7 Å). These confirmed that the affinities of the nACE-selective SG6, 33RE and ketoACE-13 are not only affected by direct interactions with the immediate environment of the binding site, but also by more distal residues. This study provides evidence for a more general mechanism of ACE inhibition involving synergistic effects of not only the S2, S1' and S2' subsites, but also residues involved in the sub-domain interface that effect the unique ways in which the two domains stabilize active site loops to favour inhibitor binding., (© 2020 The Author(s).)

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [ PSI + ]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-β structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-β structures gradually occur., Competing Interests: The authors declare no competing interest.

The modulation of structural fibrous protein and polysaccharide biopolymers for the design of biomaterials is still relatively challenging due to the non-trivial nature of the transformation from a biopolymer's native state to a more usable form. To gain insight into the nature of the molecular interaction between silk and cellulose chains, we characterized the structural, thermal and morphological properties of silk-cellulose biocomposites regenerated from the ionic liquid, 1-ethyl-3-methylimidazolium acetate (EMIMAc), as a function of increasing coagulation agent concentrations. We found that the cellulose crystallinity and crystal size are dependent on the coagulation agent, hydrogen peroxide solution. The interpretation of our results suggests that the selection of a proper coagulator is a critical step for controlling the physicochemical properties of protein-polysaccharide biocomposite materials., Competing Interests: Declaration of competing interest All authors declare no financial and/or personal relationships with any person or organization which may have inappropriately influenced the work in this study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Gap junction protein alpha 3 (GJA3), an important pathogenic gene of congenital cataracts, encodes the transmembrane protein connexin46, which functions as an intercellular channel for voltage and chemical gating by forming dodecamers. This study systematically collected nsSNP information for the GJA3 gene from SNP databases and literature and screened for nsSNPs with high risks of pathogenicity., Results: A total of 379 nsSNPs of GJA3 were identified. A total of 88 high-risk pathogenic GJA3 nsSNPs were found, including 31 published nsSNPs associated with congenital cataracts and 57 novel nsSNPs predicted by all eight online tools. The 88 high-risk pathogenic mutations, which are related to 67 amino acids in the wild-type sequences, cause a decrease in protein stability according to I-Mutant 3.0, MUpro and INPS. G2 and R33 were predicted to participate in post-translational modification and ligand binding by ModPred, RaptorX Binding and COACH. Additionally, high-risk mutations were likely to involve highly conserved sites, random coils, alpha helixes, and extracellular loops and were accompanied by changes in amino acid size, charge, hydrophobicity and spatial structure., Conclusions: Eighty-eight high-risk pathogenic nsSNPs of GJA3 were screened out in the study, 57 of which were newly reported. The combination of multiple in silico tools is highly efficient for targeting pathogenic sites.

The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a β sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the β sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dermatofibromas are common benign skin lesions, the etiology of which is poorly understood. We identified two unrelated pedigrees in which there was autosomal dominant transmission of multiple dermatofibromas. Whole exome sequencing revealed a rare shared heterozygous missense variant in the F13A1 gene encoding factor XIII subunit A (FXIII-A), a transglutaminase involved in hemostasis, wound healing, tumor growth, and apoptosis. The variant (p.Lys679Met) has an allele frequency of 0.0002 and is predicted to be a damaging mutation. Recombinant human Lys679Met FXIII-A demonstrated reduced fibrin crosslinking activity in vitro. Of note, the treatment of fibroblasts with media containing Lys679Met FXIII-A led to enhanced adhesion, proliferation, and type I collagen synthesis. Immunostaining revealed co-localization between FXIII-A and α4β1 integrins, more prominently for Lys679Met FXIII-A than the wild type. In addition, both the α4β1 inhibitors and the mutation of the FXIII-A Isoleucine-Leucine-Aspartate-Threonine (ILDT) motif prevented Lys679Met FXIII-A-dependent proliferation and collagen synthesis of fibroblasts. Our data suggest that the Lys679Met mutation may lead to a conformational change in the FXIII-A protein that enhances α4-integrin binding and provides insight into an unexpected role for FXIII-A in the pathobiology of familial dermatofibroma., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Ninety-five percent of all transmembrane proteins exist in kinetically trapped aggregation-prone states that have been directly linked to neurodegenerative diseases. Interestingly, the primary sequence almost invariably avoids off-pathway aggregate formation, by folding reliably into its native, thermodynamically stabilized structure. However, with the rising incidence of protein aggregation diseases, it is now important to understand the underlying mechanism(s) of membrane protein aggregation. Micromolecular physicochemical and biochemical alterations in the primary sequence that trigger the formation of macromolecular cross-β aggregates can be measured only through combinatorial spectroscopic experiments. Here, we developed spectroscopic thermal perturbation with 117 experimental variables to assess how subtle protein sequence variations drive the molecular transition of the folded protein to oligomeric aggregates. Using the Yersinia pestis outer transmembrane β-barrel Ail as a model, we delineated how a single-residue substitution that alters the membrane-anchoring ability of Ail significantly contributes to the kinetic component of Ail stability. We additionally observed a stabilizing role for interface aliphatics, and that interface aromatics physicochemically contribute to Ail self-assembly and aggregation. Moreover, our method identified the formation of structured oligomeric intermediates during Ail aggregation. We show that the self-aggregation tendency of Ail is offset by the evolution of a thermodynamically compromised primary sequence that balances folding, stability, and oligomerization. Our approach provides critical information on how subtle changes in protein primary sequence trigger cross-β fibril formation, with insights that have direct implications for deducing the molecular progression of neurodegeneration and amyloidogenesis in humans., (© 2020 Gupta and Mahalakshmi.)

Depending on the amino acid sequence, as well as the local environment, some peptides have the capability to fold into multiple secondary structures. Conformational switching between such structures is a key element of protein folding and aggregation. Specifically, understanding the molecular mechanism underlying the transition from an α-helix to a β-hairpin is critical because it is thought to be a harbinger of amyloid assembly. In this study, we explore the energy landscape for an 18-residue peptide (DP5), designed by Araki and Tamura to exhibit equal propensities for the α-helical and β-hairpin forms. We find that the degeneracy is encoded in the multifunnel nature of the underlying free energy landscape. In agreement with experiment, we also observe that mutation of tyrosine at position 12 to serine shifts the equilibrium in favor of the α-helix conformation, by altering the landscape topography. The transition from the α-helix to the β-hairpin is a complex stepwise process, and occurs via collapsed coil-like intermediates. Our findings suggest that even a single mutation can tune the emergent features of the landscape, providing an efficient route to protein design. Interestingly, the transition pathways for the conformational switch seem to be minimally perturbed upon mutation, suggesting that there could be universal microscopic features that are conserved among different switch-competent protein sequences.

S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) are involved in diverse cellular functions. These enzymes show little sequence conservation but have a conserved structural fold. The DNA MTases have characteristic motifs that are involved in AdoMet binding, DNA target recognition and catalysis. Motif III of these MTases have a highly conserved acidic residue, often an aspartate, whose functional significance is not clear. Here, we report a mutational study of the residue in the β family MTase of the Type III restriction-modification enzyme EcoP15I. Replacement of this residue by alanine affects its methylation activity. We propose that this residue contributes to the affinity of the enzyme for AdoMet. Analysis of the structures of DNA, RNA and protein MTases reveal that the acidic residue is conserved in all of them, and interacts with N6 of the adenine moiety of AdoMet. Interestingly, in the SET-domain protein lysine MTases, which have a fold different from other AdoMet-dependent MTases, N6 of the adenine moiety is hydrogen bonded to the main chain carbonyl group of the histidine residue of the highly conserved motif III. Our study reveals the evolutionary conservation of a carbonyl group in DNA, RNA and protein AdoMet-dependent MTases for specific interaction by hydrogen bond with AdoMet, despite the lack of overall sequence conservation.

In this study, 1064 nonhomologous "unsplit", "one-strand split" and "two-strand split" right-handed βαβ-units having standard α-helices and loops up to seven residues in length have been analyzed. It was found that the α-helices in these kinds of βαβ-units have different distributions of the hydrophobic and hydrophilic amino acid residues along the chain. In the unsplit βαβ-units, most α-helices have hydrophobic residues in positions N4-N7-N8-N11 or N6-N7-N10, where N1 is the first N-terminal residue. In the one-strand split βαβ-units, most α-helices have hydrophobic residues in positions N4-N7-N8-N11 and those in two-strand split βαβ-units in positions N4-N5-N8-N12. On the other hand, in all kinds of βαβ-units, there are commonly occurring hydrophobic stripes of type C4-C7-C8 at the C-terminal parts of the α-helices. As a rule, the C- and N-terminal hydrophobic stripes overlap and the extent of their overlapping determine the length of α-helices., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Complex amyloid aggregation of amyloid-β (1-40) (Aβ 1-40 ) in terms of monomer structures has not been fully understood. Herein, we report the microscopic mechanism and pathways of Aβ 1-40 aggregation with macroscopic viewpoints through tuning its initial structure and solubility. Partial helical structures of Aβ 1-40 induced by low solvent polarity accelerated cytotoxic Aβ 1-40 amyloid fibrillation, while predominantly helical folds did not aggregate. Changes in the solvent polarity caused a rapid formation of β-structure-rich protofibrils or oligomers via aggregation-prone helical structures. Modulation of the pH and salt concentration transformed oligomers to protofibrils, which proceeded to amyloid formation. We reveal diverse molecular mechanisms underlying Aβ 1-40 aggregation with conceptual energy diagrams and propose that aggregation-prone partial helical structures are key to inducing amyloidogenesis. We demonstrate that context-dependent protein aggregation is comprehensively understood using the macroscopic phase diagram, which provides general insights into differentiation of amyloid formation and phase separation from unfolded and folded structures.

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13 C and 15 N chemical shifts was observed for residues in the four repeats, indicating a single β-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit β-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo., Competing Interests: Conflict of interest statement: I.H., in his role as a staff scientist at the National High Magnetic Field Laboratory to assist external users, has a previous copublication with Robert Tycko.

Analyses of the hydropathic environments of protein amino acid residues reveal structural information on multiple levels. The interactions made by each residue are the basis for sidechain (rotamer) conformation and ultimately for secondary, tertiary and even quaternary protein structure. By identifying and characterizing the interactions for each residue type, we are developing a basis set of environmental data that can be used to understand protein structure. This work focuses alanine and its roles. We calculated and analyzed separately backbone-to-environment and sidechain-to-environment 3D maps for over 57,000 alanines that, with respect to hydrophobic and polar interactions, show the environment around each. After binning by backbone ϕ and ψ angles, we clustered each bin with k-means based on calculated map similarities between map-map pairs. Four bins were examined in detail: one in the β-pleat region, two in the right-hand α-helix (RHα) region and one in the left-hand α-helix region of the Ramachandran plot. All regions indicated a common map motif of hydrophobic-hydrophobic interactions along the CA-CB axis, accounting for 62% in the β-pleat bin, about one-third in the two RHα bins and 42% in the LHα bin. Another shared motif shows no interactions along the CA-CB axis; this was uncommon (8%) in β-pleat, but >30% elsewhere. The maps calculated for the two RHα bins are extremely similar (pairwise >0.9787), which suggests that the hydropathic interaction sets or motifs found around each residue are conserved. Altogether, these results are integral to a new paradigm for understanding protein structure and function., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Alzheimer's disease is associated with the abnormal self-assembly of amyloid-β (Aβ) peptide into toxic oligomers and fibrils. Recent experiments reported that Aβ16-22, containing the central hydrophobic core (CHC) of Aβ, formed antiparallel β-sheet fibrils, while its E22Q mutant self-assembled into parallel β-sheet fibrils. However, the molecular mechanisms underlying E22Q-mutation-induced parallel β-sheet fibril formation are not well understood. Herein, we performed molecular dynamics (MD) simulations to study the dimerization processes of Aβ16-22 and Aβ16-22E22Q peptides. β-Sheet dimers with diverse hydrogen bond arrangements were observed and they exhibited highly dynamic and interconverting properties. An antiparallel-to-parallel β-sheet transition occurred in the assembly process of the E22Q mutant, but not in that of Aβ16-22. During this conformational transformation process, the inter-molecular Q22-Q22 hydrogen bonds were first formed and acted as a binder to facilitate the two chains forming a parallel orientation, then the hydrophobic interactions between residues in the CHC region consolidated this arrangement and drove the main-chain H-bond formation, hence resulting in parallel β-sheet formation. However, parallel β-sheets were less populated than antiparallel β-sheets of Aβ16-22E22Q dimers. In order to explore whether parallel β-sheets became dominant in larger size oligomers, we investigated the conformational ensembles of Aβ16-22 and Aβ16-22E22Q octamers by conducting replica exchange molecular dynamics (REMD) simulations. The REMD simulations revealed that the population of parallel β-strand alignment increased with an increase of the size of ordered Aβ16-22E22Q β-sheet oligomers, implying that the formation of full parallel β-sheets requires larger sized oligomers. Our findings provide a mechanistic explanation for the E22Q-mutation-induced formation of parallel β-sheet fibrils observed experimentally.

Bacterial cell division is orchestrated by the Z ring, which is formed by single-stranded treadmilling protofilaments of FtsZ. In Streptomyces, during sporulation, multiple Z rings are assembled and lead to formation of septa that divide a filamentous hyphal cell into tens of prespore compartments. We describe here mutant alleles of ftsZ in Streptomyces coelicolor and Streptomyces venezuelae that perturb cell division in such a way that constriction is initiated along irregular spiral-shaped paths rather than as regular septa perpendicular to the cell length axis. This conspicuous phenotype is caused by amino acid substitutions F37I and F37R in β strand S2 of FtsZ. The F37I mutation leads, instead of regular Z rings, to formation of relatively stable spiral-shaped FtsZ structures that are capable of initiating cell constriction. Further, we show that the F37 mutations affect the polymerization properties and impair the cooperativity of FtsZ assembly in vitro. The results suggest that specific residues in β strand S2 of FtsZ affect the conformational switch in FtsZ that underlies assembly cooperativity and enable treadmilling of protofilaments, and that these features are required for formation of regular Z rings. However, the data also indicate FtsZ-directed cell constriction is not dependent on assembly cooperativity., (© 2019 John Wiley & Sons Ltd.)

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of β-PFTs (aβ-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double β-barrel, a common feature of the aβ-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.

Many protein and peptide sequences are self-assembled into β-sheet-rich fibrous structures called amyloids. Their atomic details provide insights into fundamental knowledge related to amyloid diseases. To study the detailed structure of the amyloid, we have developed a model system that mimics the self-assembling process of the amyloid within a water-soluble protein, termed peptide self-assembly mimic (PSAM). PSAM enables capturing of a peptide sequence within a water-soluble protein, thus making structural and energetics-related studies possible. In this work, we extend our PSAM approach to a naturally occurring chameleon sequence from αB crystallin. We chose "Val-Leu-Gly-Asp-Val (VLGDV)", a five amino-acid sequence, which forms a β-turn in the native structure and a β-barrel in the amyloid oligomer cylindrin, as a grafting sequence to the PSAM scaffold. The crystal structure revealed that the sequence grafting induced β-sheet bending at the grafted site. We further investigated the role of the central glycine residue and found that its role in the β-sheet bending is dependent on the neighboring residues. The ability of PSAM to observe the structural alterations induced by the grafted sequence provides an opportunity to evaluate the structural impact of a sequence from the peptide self-assembly., (© 2019 Wiley Periodicals, Inc.)

Understanding the initial events of aggregation of amyloid β monomers to form β-sheet rich fibrils is useful for the development of therapeutics for Alzheimer's disease. In this context, the changes in energetics involved in the aggregation of helical amyloid β monomers into β-sheet rich dimers have been investigated using umbrella sampling simulations and density functional theory calculations. The results from umbrella sampling simulations for the free energy profile for the interconversion closely agree with the results of density functional theory calculations. The results reveal that helical peptides converted to β-sheet structures through coil-like conformations as intermediates that are mostly stabilized by intramolecular hydrogen bonds. The stabilization of intermediate structures could be a possible way to inhibit fibril formation. Mutations substantially decrease the height of the energy barrier for interconversion from α-helix to β-sheet structure when compared to that of the wild type, something that is attributed to an increase in the number of intramolecular hydrogen bonds between backbone atoms in the coil structures that correspond to a maximum value on the free energy surface. The reduction of the energy barrier leads to an enhancement of the rate of aggregation of amyloid β monomers upon introduction of various familial mutations, which is consistent with previous experimental reports.

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of β-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mammalian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.

In order to investigate the early phase of the amyloid formation by the short amyloidogenic octapeptide sequence ('NFGAILSS') derived from IAPP, we carried out a 100ns all-atom molecular dynamics (MD) simulations of systems that contain 27 peptides and over 30,000 water molecules. The large-scale calculations were performed for the wild type sequence and seven alanine-scanned sequences using AMBER 8.0 on RIKEN's special purpose MD-GRAPE3 supercomputer, using the all-atom point charge force field ff99, which do not favor β-structures. Large peptide clusters (size 18-26 mers) were observed for all simulations, and our calculations indicated that isoleucine at position 5 played important role in the formation of β-rich clusters. In the oligomeric state, the wild type and the S7A sequences had the highest β-structure content (~14%), as calculated by DSSP, in line with experimental observations, whereas I5A and G3A had the highest helical content (~20%). Importantly, the β-structure preferences of wild type IAPP originate from its association into clusters and are not intrinsic to its sequence. Altogether, the results of this first large-scale, multi-peptide all-atom molecular dynamics simulation appear to provide insights into the mechanism of amyloidogenic and non-amyloidogenic oligomers that mainly corroborate previous experimental observations.

Proteins and peptides play a predominant role in biochemical reactions of living cells. In these complex environments, not only the constitution of the molecules but also their three-dimensional configuration defines their functionality. This so-called secondary structure of proteins is crucial for understanding their function in living matter. Misfolding, for example, is suspected as the cause of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Ultimately, it is necessary to study a single protein and its folding dynamics. Here, we report a first step in this direction, namely ultrasensitive detection and discrimination of in vitro polypeptide folding and unfolding processes using resonant plasmonic nanoantennas for surface-enhanced vibrational spectroscopy. We utilize poly-l-lysine as a model system which has been functionalized on the gold surface. By in vitro infrared spectroscopy of a single molecular monolayer at the amide I vibrations we directly monitor the reversible conformational changes between α-helix and β-sheet states induced by controlled external chemical stimuli. Our scheme in combination with advanced positioning of the peptides and proteins and more brilliant light sources is highly promising for ultrasensitive in vitro studies down to the single protein level.

βαβ super-secondary structures constitute the basic building blocks of (β/α) 8 class of proteins. Despite the success in designing super-secondary structures, till date, there is not a single example of a natural βαβ sequence known to fold in isolation. In this chapter, to address the finding the "needles" in the haystack scenario, we have combined the sequence preferences and structural features of independent βαβ motifs, dictated by natural selection, with rationally derived parameters from a designed βαβ motif adopting stable fold in solution. Guided by this approach, a set of potential βαβ sequences from (β/α) 8 /TIM barrels are proposed as likely candidates for autonomously folding based on the assessment of their foldability.

The 2-hydroxyglutaric aciduria (2-HGA) is a rare neurometabolic disorder that leads to the development of brain damage. It is classified into three categories: D-2-HGA, L-2-HGA, and combined D,L-2-HGA. The D-2-HGA includes two subtypes: type I and type II caused by the mutations in D2HGDH and IDH2 proteins, respectively. In this study, we studied six mutations, four in the D2HGDH (I147S, D375Y, N439D, and V444A) and two in the IDH2 proteins (R140G, R140Q). We performed in silico analysis to investigate the pathogenicity and stability changes of the mutant proteins using pathogenicity (PANTHER, PhD-SNP, SIFT, SNAP, and META-SNP) and stability (i-Mutant, MUpro, and iStable) predictors. All the mutations of both D2HGDH and IDH2 proteins were predicted as disease causing except V444A, which was predicted as neutral by SIFT. All the mutants were also predicted to be destabilizing the protein except the mutants D375Y and N439D. DSSP plugin of the PyMOL and Molecular Dynamics Simulations (MDS) were used to study the structural changes in the mutant proteins. In the case of D2HGDH protein, the mutations I147S and V444A that are positioned in the beta sheet region exhibited higher Root Mean Square Deviation (RMSD), decrease in compactness and number of intramolecular hydrogen bonds compared to the mutations N439D and D375Y that are positioned in the turn and loop region, respectively. While the mutants R140Q and R140QG that are positioned in the alpha helix region of the protein. MDS results revealed the mutation R140Q to be more destabilizing (higher RMSD values, decrease in compactness and number of intramolecular hydrogen bonds) compared to the mutation R140G of the IDH2 protein. This study is expected to serve as a platform for drug development against 2-HGA and pave the way for more accurate variant assessment and classification for patients with genetic diseases.

The human stomach pathogen Helicobacter pyloriattaches to healthy and inflamed gastric tissue through members of a paralogous family of 'Helicobacter outer membrane proteins' (Hops), including adhesins BabA, SabA, HopQ, LabA and HopZ. Hops share a conserved 25 kDa C-terminal region that is thought to form an autotransporter-like transmembrane domain. Instead, our results show that Hops contain a non-continuous transmembrane domain, composed of seven predicted β-strands at the C-terminus and one at the N-terminus. Folding and outer membrane localization of the C-terminal β-domain critically depends on a predicted transmembrane β-strand within the first 16 N-terminal residues. The N-terminus is shown to reside in the periplasm, and our crystal and small angle X-ray scattering structures for the SabA extracellular domain reveal a conserved coiled-coil stem domain that connects to transmembrane β-strand 1 and 2. Taken together, our data show that Hop adhesins represent a novel outer membrane protein topology encompassing an OmpA-like 8-stranded β-barrel that is interrupted by a 15-108 kDa domain inserted inside the first extracellular loop. The insertion of large, folded domains in an extracellular loop is unprecedented in bacterial outer membrane proteins and is expected to have important consequences on how these proteins reach the cell surface., (© 2018 John Wiley & Sons Ltd.)

Spider venoms are complex mixtures of proteins, peptides and small organic and inorganic molecules. Among the proteins, phospholipases D (PLDs) present the major portion, and till now they are the most studied enzymes in spider venom. These PLDs have been divided into two classes, I and II, based on their primary and tertiary structure. Currently, crystal structures of both classes of these enzymes are available in the Protein Data Bank (PDB). Their three-dimensional structure is composed of eight α-helices and eight β-strands forming the ubiquitous fold called triosephosphate isomerase (TIM) barrel. These enzymes use general acid-base catalysis to hydrolyzes their substrate. In this review, we have described the structural features, structure-based mechanisms of catalysis, maturation, and inhibition of these enzymes using the synthetic inhibitor., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The sensor histidine kinases of two-component signal-transduction systems (TCSs) are essential for bacteria to adapt to variable environmental conditions. The two-component regulatory system BaeS/R increases multidrug and metal resistance in Salmonella and Escherichia coli. In this study, we report the X-ray structure of the periplasmic sensor domain of BaeS from Serratia marcescens FS14. The BaeS sensor domain (34-160) adopts a mixed α/β-fold containing a central four-stranded antiparallel β-sheet flanked by a long N-terminal α-helix and additional loops and a short C-terminal α-helix on each side. Structural comparisons revealed that it belongs to the PDC family with a remarkable difference in the orientation of the helix α2. In the BaeS sensor domain, this helix is situated perpendicular to the long helix α1 and holds helix α1 in the middle with the beta sheet, whereas in other PDC domains, helix α2 is parallel to helix α1. Because the helices α1 and α2 is involved in the dimeric interface, this difference implies that BaeS uses a different dimeric interface compared with other PDC domains. Proteins 2017; 85:1784-1790. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)