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β-Structure-rich amyloid fibrils are hallmarks of several diseases, including Alzheimer’s (AD), Parkinson’s (PD), and type 2 diabetes (T2D). While amyloid fibrils typically consist of parallel β-sheets, the anti-parallel β-hairpin is a structural motif accessible to amyloidogenic proteins in their monomeric and oligomeric states. Here, to investigate implications of β-hairpins in amyloid formation, potential β-hairpin-forming amyloidogenic segments in the human proteome were predicted based on sequence similarity with β-hairpins previously observed in Aβ, α-synuclein, and islet amyloid polypeptide, amyloidogenic proteins associated with AD, PD, and T2D, respectively. These three β-hairpins, established upon binding to the engineered binding protein β-wrapin AS10, are characterized by proximity of two sequence segments rich in hydrophobic and aromatic amino acids, with high β-aggregation scores according to the TANGO algorithm. Using these criteria, 2505 potential β-hairpin-forming amyloidogenic segments in 2098 human proteins were identified. Characterization of a test set of eight protein segments showed that seven assembled into Thioflavin T-positive aggregates and four formed β-hairpins in complex with AS10 according to NMR. One of those is a segment of prostatic acid phosphatase (PAP) comprising amino acids 185–208. PAP is naturally cleaved into fragments, including PAP(248−286) which forms functional amyloid in semen. We find that PAP(185−208) strongly decreases the protein concentrations required for fibril formation of PAP(248−286) and of another semen amyloid peptide, SEM1(86−107), indicating that it promotes nucleation of semen amyloids. In conclusion, β-hairpin-forming amyloidogenic protein segments could be identified in the human proteome with potential roles in functional or disease-related amyloid formation.

Climate change has been associated with outbreaks of mycotoxicosis following periods of drought, enhanced fungal growth, and increased exposure to mycotoxins. For detoxification, the inclusion of clay-based materials in food and drinking water has resulted in a very promising strategy to reduce mycotoxin exposure. In this strategy, mycotoxins are tightly sorbed to high-affinity clay particles in the gastrointestinal tract, thus decreasing bioavailability, uptake to blood, and potential toxicity. This study investigated the ability of chlorophyll and chlorophyllin-amended montmorillonite clays to decrease the toxicity of ochratoxin A (OTA). The sorption mechanisms of OTA binding to surfaces of sorbents, as well as binding parameters such as capacity, affinity, enthalpy, and free energy, were examined. Chlorophyll-amended organoclay (CMCH) demonstrated the highest binding (72%) and was better than the chlorophyllin-amended hydrophilic clay (59%), possibly due to the hydrophobicity of OTA (LogP 4.7). In silico studies using molecular dynamics simulations showed that CMCH improves OTA binding in comparison to parent clay in line with experiments. Simulations depicted that chlorophyll amendments on clay facilitated OTA molecules binding both directly, through enhancing OTA binding on the clay, or predominantly indirectly, through OTA molecules interacting with bound chlorophyll amendments. Simulations uncovered the key role of calcium ions in OTA binding, particularly in neutral conditions, and demonstrated that CMCH binding to OTA is enhanced under both neutral and acidic conditions. Furthermore, the protection of various sorbents against OTA-induced toxicity was carried out using two living organisms (Hydra vulgaris and Caenorhabditis elegans) which are susceptible to OTA toxicity. This study showed the significant detoxification of OTA (33% to 100%) by inclusion of sorbents. Organoclay (CMCH) at 0.5% offered complete protection. These findings suggest that the chlorophyll-amended organoclays described in this study could be included in food and feed as OTA binders and as potential filter materials for water and beverages to protect against OTA contaminants during outbreaks and emergencies.

To date, over 150 chemical modifications to the four canonical RNA bases have been discovered, known collectively as the epitranscriptome. Many of these modifications have been implicated in a variety of cellular processes and disease states. Additional work has been done to identify proteins known as “readers” that selectively interact with RNAs that contain specific chemical modifications. Protein interactomes with N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytosine (m5C), and 8-oxo-7,8-dihydroguanosine (8-oxoG) have been determined, mainly through experimental advances in proteomics techniques. However, relatively few proteins have been confirmed to bind directly to RNA containing these modifications. Furthermore, for many of these protein readers, the exact binding mechanisms as well as the exclusivity for recognition of modified RNA species remain elusive, leading to questions regarding their roles within different cellular processes. In the case of the YT-521B homology (YTH) family of proteins, both experimental and in silico techniques have been leveraged to provide valuable biophysical insights into the mechanisms of m6A recognition at atomic resolution. To date, the YTH family is one of the best characterized classes of readers. Here, we review current knowledge about epitranscriptome recognition of the YTH domain proteins from previously published experimental and computational studies. We additionally outline knowledge gaps for proteins beyond the well-studied human YTH domains and the current in silico techniques and resources that can enable investigation of protein interactions with modified RNA outside of the YTH-m6A context.

Every two years groups worldwide participate in the Critical Assessment of Protein Structure Prediction (CASP) experiment to blindly test the strengths and weaknesses of their computational methods. CASP has significantly advanced the field but many hurdles still remain, which may require new ideas and collaborations. In 2012 a web-based effort called WeFold, was initiated to promote collaboration within the CASP community and attract researchers from other fields to contribute new ideas to CASP. Members of the WeFold coopetition (cooperation and competition) participated in CASP as individual teams, but also shared components of their methods to create hybrid pipelines and actively contributed to this effort. We assert that the scale and diversity of integrative prediction pipelines could not have been achieved by any individual lab or even by any collaboration among a few partners. The models contributed by the participating groups and generated by the pipelines are publicly available at the WeFold website providing a wealth of data that remains to be tapped. Here, we analyze the results of the 2014 and 2016 pipelines showing improvements according to the CASP assessment as well as areas that require further adjustments and research.

1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) is a bacterial-derived metabolite that binds the aryl hydrocarbon receptor (AhR) and exhibits anti-inflammatory activity in the gut. The structure-dependent AhR activity of hydroxyl/carboxy-substituted naphthoic acids (NAs) was determined in young adult mouse colonic (YAMC) cells and human Caco2 colon cancer cells using CYP1A1/CYP1B1 mRNAs as Ah-responsive genes. Compounds used in this study include 1,4-, 3,5-, and 3,7-DHNA, 1,4-dimethoxy-2-naphthoic acid (1,4-DMNA), 1- and 4-hydroxy-2-naphthoic acid (1-HNA, 4-HNA), 1- and 2-naphthoic acid (1-NA, 2-NA), and 1- and 2-naphthol (1-NOH, 2-NOH). 1,4-DHNA was the most potent compound among hydroxyl/carboxy naphthalene derivatives, and the fold induction response for CYP1A1 and CYP1B1 was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in YAMC and Caco2 cells. 1- and 4-HNA were less potent than 1,4-DHNA but induced maximal (TCDD-like) response for CYP1B1 (both cell lines) and CYP1A1 (Caco2 cells). With the exception of 1- and 2-NA, all compounds significantly induced Cyp1b1 in YAMC cells and these responses were not observed in AhR-deficient YAMC cells generated using CRISPR/Cas9 technology. In addition, we also observed that 1- and 2-NOH (and 1,4-DHNA) were weak AhR agonists, and 1- and 2-NOH also exhibited partial AhR antagonist activity. Structure-activity relationship studies for CYP1A1 but not CYP1B1 were similar in both cell lines, and CYP1A1 induction required one or both 1,4-dihydroxy substituents and activity was significantly enhanced by the 2-carboxyl group. We also used computational analysis to show that 1,4-DHNA and TCDD share similar interactions within the AhR binding pocket and differ primarily due to the negatively charged group of 1,4-DHNA.

The oxidation of RNA has been implicated in the development of many diseases. Among the four ribonucleotides, guanosine is the most susceptible to oxidation, resulting in the formation of 8-oxo-7,8-dihydroguanosine (8-oxoG). Despite the limited knowledge about how cells regulate the detrimental effects of oxidized RNA, cellular factors involved in its regulation have begun to be identified. One of these factors is polynucleotide phosphorylase (PNPase), a multifunctional enzyme implicated in RNA turnover. In the present study, we have examined the interaction of PNPase with 8-oxoG in atomic detail to provide insights into the mechanism of 8-oxoG discrimination. We hypothesized that PNPase subunits cooperate to form a binding site using the dynamic SFF loop within the central channel of the PNPase homotrimer. We evolved this site using a novel approach that initially screened mutants from a library of beneficial mutations and assessed their interactions using multi-nanosecond Molecular Dynamics simulations. We found that evolving this single site resulted in a fold change increase in 8-oxoG affinity between 1.2 and 1.5 and/or selectivity between 1.5 and 1.9. In addition to the improvement in 8-oxoG binding, complementation of K12 Δpnp with plasmids expressing mutant PNPases caused increased cell tolerance to H2O2. This observation provides a clear link between molecular discrimination of RNA oxidation and cell survival. Moreover, this study provides a framework for the manipulation of modified-RNA protein readers, which has potential application in synthetic biology and epitranscriptomics.

Compstatin peptides are complement inhibitors that bind and inhibit cleavage of complement C3. Peptide binding is enhanced by hydrophobic interactions; however, poor solubility promotes aggregation in aqueous environments. We have designed new compstatin peptides derived from the W4A9 sequence (Ac-ICVWQDWGAHRCT-NH2, cyclized between C2 and C12), based on structural, computational, and experimental studies. Furthermore, we developed and utilized a computational framework for the design of peptides containing non-natural amino acids. These new compstatin peptides contain polar N-terminal extensions and non-natural amino acid substitutions at positions 4 and 9. Peptides with α-modified non-natural alanine analogs at position 9, as well as peptides containing only N-terminal polar extensions, exhibited similar activity compared to W4A9, as quantified via ELISA, hemolytic, and cell-based assays, and showed improved solubility, as measured by UV absorbance and reverse-phase HPLC experiments. Because of their potency and solubility, these peptides are promising candidates for therapeutic development in numerous complement-mediated diseases.

BackgroundThe complement protein C5a acts by primarily binding and activating the G-protein coupled C5a receptor C5aR (CD88), and is implicated in many inflammatory diseases. The cyclic hexapeptide PMX53 (sequence Ace-Phe-[Orn-Pro-dCha-Trp-Arg]) is a full C5aR antagonist of nanomolar potency, and is widely used to study C5aR function in disease.ResultsWe construct for the first time molecular models for the C5aR:PMX53 complex without the a priori use of experimental constraints, via a computational framework of molecular dynamics (MD) simulations, docking, conformational clustering and free energy filtering. The models agree with experimental data, and are used to propose important intermolecular interactions contributing to binding, and to develop a hypothesis for the mechanism of PMX53 antagonism.ConclusionThis work forms the basis for the design of improved C5aR antagonists, as well as for atomic-detail mechanistic studies of complement activation and function. Our computational framework can be widely used to develop GPCR-ligand structural models in membrane environments, peptidomimetics and other chemical compounds with potential clinical use.

The V3 loop of the glycoprotein 120 (gp120) is a contact point for cell entry of HIV-1 leading to infection. Despite sequence variability and lack of specific structure, the highly flexible V3 loop possesses a well-defined role in recognizing and selecting cell-bound coreceptors CCR5 and CXCR4 through a mechanism of charge complementarity. We have performed two independent molecular dynamics (MD) simulations to gain insights into the dynamic character of two V3 loops with slightly different sequences, but significantly different starting crystallographic structures. We have identified highly populated trajectory-specific salt bridges between oppositely charged stem residues Arg9 and Glu25 or Asp29. The two trajectories share nearly identical correlated motions within the simulations, despite their different overall structures. High occupancy salt bridges play a key role in the major cross-correlated motions in both trajectories, and may be responsible for transient structural stability in preparation for coreceptor binding. In addition, the two V3 loops visit conformations with similarities in spatial distributions of electrostatic potentials, despite their inherent flexibility, which may play a role in coreceptor recognition. It is plausible that cooperativity between overall electrostatic potential, charged residue interactions, and correlated motions could be associated with a coreceptor selection and binding.

The V3 loop of the glycoprotein 120 (gp120) is a contact point for cell entry of HIV-1 leading to infection. Despite sequence variability and lack of specific structure, the highly flexible V3 loop possesses a well-defined role in recognizing and selecting cell-bound coreceptors CCR5 and CXCR4 through a mechanism of charge complementarity. We have performed two independent molecular dynamics (MD) simulations to gain insights into the dynamic character of two V3 loops with slightly different sequences, but significantly different starting crystallographic structures. We have identified highly populated trajectory-specific salt bridges between oppositely charged stem residues Arg9 and Glu25 or Asp29. The two trajectories share nearly identical correlated motions within the simulations, despite their different overall structures. High occupancy salt bridges play a key role in the major cross-correlated motions in both trajectories, and may be responsible for transient structural stability in preparation for coreceptor binding. In addition, the two V3 loops visit conformations with similarities in spatial distributions of electrostatic potentials, despite their inherent flexibility, which may play a role in coreceptor recognition. It is plausible that cooperativity between overall electrostatic potential, charged residue interactions, and correlated motions could be associated with a coreceptor selection and binding.

Abstract Every two years groups worldwide participate in the Critical Assessment of Protein Structure Prediction (CASP) experiment to blindly test the strengths and weaknesses of their computational methods. CASP has significantly advanced the field but many hurdles still remain, which may require new ideas and collaborations. In 2012 a web-based effort called WeFold, was initiated to promote collaboration within the CASP community and attract researchers from other fields to contribute new ideas to CASP. Members of the WeFold coopetition (cooperation and competition) participated in CASP as individual teams, but also shared components of their methods to create hybrid pipelines and actively contributed to this effort. We assert that the scale and diversity of integrative prediction pipelines could not have been achieved by any individual lab or even by any collaboration among a few partners. The models contributed by the participating groups and generated by the pipelines are publicly available at the WeFold website providing a wealth of data that remains to be tapped. Here, we analyze the results of the 2014 and 2016 pipelines showing improvements according to the CASP assessment as well as areas that require further adjustments and research.

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan receptor and member of the nuclear receptor superfamily. Among a series of methylene substituted diindolylmethanes (C-DIMs) containing substituted phenyl and heteroaromatic groups, we identified 1,1-bis(3′-indolyl)-1-(4-pyridyl)-methane (DIM-C-Pyr-4) as an activator of COUP-TFI. Structure activity studies with structurally diverse heteroaromatic C-DIMs showed that the pyridyl substituted compound was active and the 4-pyridyl substituent was more potent than the 2- or 3-pyridyl analogs in transactivation assays in breast cancer cells. The DIM-C-Pyr-4 activated chimeric GAL4-COUP-TFI constructs containing full length, C- or N-terminal deletions, and transactivation was inhibited by phosphatidylinositol-3-kinase and protein kinase A inhibitors. However, DIM-C-Pyr-4 also induced transactivation and interactions of COUP-TFI and steroid receptor coactivators-1 and -2 in mammalian two-hybrid assays, and ligand-induced interactions of the C-terminal region of COUP-TFI were not affected by kinase inhibitors. We also showed that DIM-C-Pyr-4 activated COUP-TFI-dependent early growth response 1 (Egr-1) expression and this response primarily involved COUP-TFI interactions with Sp3 and to a lesser extent Sp1 bound to the proximal region of the Egr-1 promoter. Modeling studies showed interactions of DIM-C-Pyr-4 within the ligand binding domain of COUP-TFI. This report is the first to identify a COUP-TFI agonist and demonstrate activation of COUP-TFI-dependent Egr-1 expression.

HIV-1 entry into host cells is mediated by interactions between the V3-loop of viral glycoprotein gp120 and chemokine receptor CCR5 or CXCR4, collectively known as HIV-1 coreceptors. Accurate genotypic prediction of coreceptor usage is of significant clinical interest and determination of the factors driving tropism has been the focus of extensive study. We have developed a method based on nonlinear support vector machines to elucidate the interacting residue pairs driving coreceptor usage and provide highly accurate coreceptor usage predictions. Our models utilize centroid-centroid interaction energies from computationally derived structures of the V3-loop:coreceptor complexes as primary features, while additional features based on established rules regarding V3-loop sequences are also investigated. We tested our method on 2455 V3-loop sequences of various lengths and subtypes, and produce a median area under the receiver operator curve of 0.977 based on 500 runs of 10-fold cross validation. Our study is the first to elucidate a small set of specific interacting residue pairs between the V3-loop and coreceptors capable of predicting coreceptor usage with high accuracy across major HIV-1 subtypes. The developed method has been implemented as a web tool named CRUSH, CoReceptor USage prediction for HIV-1, which is available at http://ares.tamu.edu/CRUSH/.

The binding of protein HIV-1 gp120 to coreceptors CCR5 or CXCR4 is a key step of the HIV-1 entry to the host cell, and is predominantly mediated through the V3 loop fragment of HIV-1 gp120. In the present work, we delineate the molecular recognition of chemokine receptor CCR5 by a dual tropic HIV-1 gp120 V3 loop, using a comprehensive set of computational tools predominantly based on molecular dynamics simulations and free energy calculations. We report, what is to our knowledge, the first complete HIV-1 gp120 V3 loop : CCR5 complex structure, which includes the whole V3 loop and the N-terminus of CCR5, and exhibits exceptional agreement with previous experimental findings. The computationally derived structure sheds light into the functional role of HIV-1 gp120 V3 loop and CCR5 residues associated with the HIV-1 coreceptor activity, and provides insights into the HIV-1 coreceptor selectivity and the blocking mechanism of HIV-1 gp120 by maraviroc. By comparing the binding of the specific dual tropic HIV-1 gp120 V3 loop with CCR5 and CXCR4, we observe that the HIV-1 gp120 V3 loop residues 13-21, which include the tip, share nearly identical structural and energetic properties in complex with both coreceptors. This result paves the way for the design of dual CCR5/CXCR4 targeted peptides as novel potential anti-AIDS therapeutics.

The V3 loop of the glycoprotein 120 (gp120) is a contact point for cell entry of HIV-1 leading to infection. Despite sequence variability and lack of specific structure, the highly flexible V3 loop possesses a well-defined role in recognizing and selecting cell-bound coreceptors CCR5 and CXCR4 through a mechanism of charge complementarity. We have performed two independent molecular dynamics (MD) simulations to gain insights into the dynamic character of two V3 loops with slightly different sequences, but significantly different starting crystallographic structures. We have identified highly populated trajectory-specific salt bridges between oppositely charged stem residues Arg9 and Glu25 or Asp29. The two trajectories share nearly identical correlated motions within the simulations, despite their different overall structures. High occupancy salt bridges play a key role in the major cross-correlated motions in both trajectories, and may be responsible for transient structural stability in preparation for coreceptor binding. In addition, the two V3 loops visit conformations with similarities in spatial distributions of electrostatic potentials, despite their inherent flexibility, which may play a role in coreceptor recognition. It is plausible that cooperativity between overall electrostatic potential, charged residue interactions, and correlated motions could be associated with a coreceptor selection and binding.

Benzene is a carcinogenic volatile organic compound (VOC) that is ubiquitously detected in enclosed spaces due to emissions from cooking activities, building materials, and cleaning products. To remove benzene and other VOCs from indoor air and protect public health, traditional fabric filters have been modified to contain activated carbons to enhance the filtration efficacy. In this study, composites derived from natural clay minerals and activated carbon were individually green-engineered with chlorophylls and were attached to the surface of filter materials. These systems were assessed for their adsorption of benzene from air using in vitro and in silico methods. Isothermal, thermodynamic, and kinetic experiments indicated that all green-engineered composites had improved binding profiles for benzene, as demonstrated by increased binding affinities (K f ≥ 900 vs 472) and lower values of Gibbs free energy (ΔG = −16.8 vs −15.2) compared to activated carbon. Adsorption of benzene to all composites was achieved quickly (< 30 min), and the green-engineered composites also showed low levels of desorption (≤ 25%). While free chlorophyll is known to be photosensitive, chlorophylls in the green-engineered composites showed photostability and maintained high binding rates (≥ 70%). Additionally, the in silico simulations demonstrated the significant contribution of chlorophyll for the overall binding of benzene in clay systems and that chlorophyll could contribute to benzene binding in the carbon-based systems. Together, these studies indicated that novel, green-engineered composite materials can be effective filter sorbents to enhance the removal of benzene from air. [Display omitted] • Green-engineered composite materials were developed to capture benzene from air. • Green-engineered materials had more favorable binding profiles for benzene. • Simulations indicated physical mechanisms were important to the overall adsorption. • These materials were photostable and had low levels of desorption. [ABSTRACT FROM AUTHOR]