Targeting the Structural Maturation Pathway of HIV-1 Reverse Transcriptase.

Formation of active HIV-1 reverse transcriptase (RT) proceeds via a structural maturation process that involves subdomain rearrangements and formation of an asymmetric p66/p66' homodimer. These studies were undertaken to evaluate whether the information about this maturation process can be used to identify small molecule ligands that retard or interfere with the steps involved. We utilized the isolated polymerase domain, p51, rather than p66, since the initial subdomain rearrangements are largely limited to this domain.

The translational repressor Glorund uses interchangeable RNA recognition domains to recognize Drosophila nanos.

The Drosophila melanogaster protein Glorund (Glo) represses nanos (nos) translation and uses its quasi-RNA recognition motifs (qRRMs) to recognize both G-tract and structured UA-rich motifs within the nos translational control element (TCE). We showed previously that each of the three qRRMs is multifunctional, capable of binding to G-tract and UA-rich motifs, yet if and how the qRRMs combine to recognize the nos TCE remained unclear. Here we determined solution structures of a nos TCEI_III RNA containing the G-tract and UA-rich motifs.

Structural and ligand binding analysis of the pet allergens Can f 1 and Fel d 7.

Introduction: Pet lipocalins are respiratory allergens with a central hydrophobic ligand-binding cavity called a calyx. Molecules carried in the calyx by allergens are suggested to influence allergenicity, but little is known about the native ligands.

Methods: To provide more information on prospective ligands, we report crystal structures, NMR, molecular dynamics, and florescence studies of a dog lipocalin allergen Can f 1 and its closely related (and cross-reactive) cat allergen Fel d 7.

Structure and IgE Cross-Reactivity among Cashew, Pistachio, Walnut, and Peanut Vicilin-Buried Peptides.

Peanut and tree-nut allergies are frequently comorbid for reasons not completely understood. Vicilin-buried peptides (VBPs) are an emerging family of food allergens whose conserved structural fold could mediate peanut/tree-nut co-allergy. Peptide microarrays were used to identify immunoglobulin E (IgE) epitopes from the N-terminus of the vicilin allergens Ara h 1, Ana o 1, Jug r 2, and Pis v 3 using serum from three patient diagnosis groups: monoallergic to either peanuts or cashew/pistachio, or dual allergic.

Nanobody Paratope Ensembles in Solution Characterized by MD Simulations and NMR.

Variable domains of camelid antibodies (so-called nanobodies or VH) are the smallest antibody fragments that retain complete functionality and therapeutic potential. Understanding of the nanobody-binding interface has become a pre-requisite for rational antibody design and engineering. The nanobody-binding interface consists of up to three hypervariable loops, known as the CDR loops.

Structure, Immunogenicity, and IgE Cross-Reactivity among Walnut and Peanut Vicilin-Buried Peptides.

Vicilin-buried peptides (VBPs) from edible plants are derived from the N-terminal leader sequences (LSs) of seed storage proteins. VBPs are defined by a common α-hairpin fold mediated by conserved CxxxCxCxxxC motifs. Here, peanut and walnut VBPs were characterized as potential mediators of both peanut/walnut allergenicity and cross-reactivity despite their low (∼17%) sequence identity.

The mosquito protein AEG12 displays both cytolytic and antiviral properties via a common lipid transfer mechanism.

The mosquito protein AEG12 is up-regulated in response to blood meals and flavivirus infection though its function remained elusive. Here, we determine the three-dimensional structure of AEG12 and describe the binding specificity of acyl-chain ligands within its large central hydrophobic cavity. We show that AEG12 displays hemolytic and cytolytic activity by selectively delivering unsaturated fatty acid cargoes into phosphatidylcholine-rich lipid bilayers.

Mapping Human Monoclonal IgE Epitopes on the Major Dust Mite Allergen Der p 2.

IgE Abs drive the symptoms of allergic disease upon cross-linking allergens on mast cells or basophils. If the IgE binding sites on the allergens could be identified, it may be useful for creating new forms of immunotherapy. However, direct knowledge of the human IgE (hIgE) epitopes is limited because of the very low frequency of IgE-producing B cells in blood.

Comparison of phytochemical composition of Ginkgo biloba extracts using a combination of non-targeted and targeted analytical approaches.

Ginkgo biloba extract (GbE) is a dietary supplement derived from an ethanolic extract of Ginkgo biloba leaves. Unfinished bulk GbE is used to make finished products that are sold as dietary supplements. The variable, complex composition of GbE makes it difficult to obtain consistent toxicological assessments of potential risk.

The Structural Basis for Nonsteroidal Anti-Inflammatory Drug Inhibition of Human Dihydrofolate Reductase.

Although nonsteroidal anti-inflammatory drugs (NSAIDs) target primarily cyclooxygenase enzymes, a subset of NSAIDs containing carboxylate groups also has been reported to competitively inhibit dihydrofolate reductase (DHFR). In this study, we have characterized NSAID interactions with human DHFR based on kinetic, NMR, and X-ray crystallographic methods. The NSAIDs target a region of the folate binding site that interacts with the -aminobenzoyl-l-glutamate (pABG) moiety of folate and inhibit cooperatively with ligands that target the adjacent pteridine-recognition subsite.

Hydrophobic ligands influence the structure, stability, and processing of the major cockroach allergen Bla g 1.

The cockroach allergen Bla g 1 forms a novel fold consisting of 12 amphipathic alpha-helices enclosing an exceptionally large hydrophobic cavity which was previously demonstrated to bind a variety of lipids. Since lipid-dependent immunoactivity is observed in numerous allergens, understanding the structural basis of this interaction could yield insights into the molecular determinants of allergenicity. Here, we report atomic modelling of Bla g 1 bound to both fatty-acid and phospholipids ligands, with 8 acyl chains suggested to represent full stoichiometric binding.

A Human IgE Antibody Binding Site on Der p 2 for the Design of a Recombinant Allergen for Immunotherapy.

Der p 2 is one of the most important allergens from the house dust mite Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment.

Transitions in DNA polymerase β μs-ms dynamics related to substrate binding and catalysis.

DNA polymerase β (pol β) plays a central role in the DNA base excision repair pathway and also serves as an important model polymerase. Dynamic characterization of pol β from methyl-TROSY 13C-1H multiple quantum CPMG relaxation dispersion experiments of Ile and Met sidechains and previous backbone relaxation dispersion measurements, reveals transitions in μs-ms dynamics in response to highly variable substrates. Recognition of a 1-nt-gapped DNA substrate is accompanied by significant backbone and sidechain motion in the lyase domain and the DNA binding subdomain of the polymerase domain, that may help to facilitate binding of the apoenzyme to the segments of the DNA upstream and downstream from the gap.

Characterization of the APLF FHA-XRCC1 phosphopeptide interaction and its structural and functional implications.

Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHA-FBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr.

Identification of drivers for the metamorphic transition of HIV-1 reverse transcriptase.

Recent structural characterizations of the p51 and p66 monomers have established an important starting point for understanding the maturation pathway of the human immunodeficiency virus (HIV)-1 reverse transcriptase p66/p51 heterodimer. This process requires a metamorphic transition of the polymerase domain leading to formation of a p66/p66' homodimer that exists as a structural heterodimer. To better understand the drivers for this metamorphic transition, we have performed NMR studies of N-labeled RT216 - a construct that includes the fingers and most of the palm domains.

A metabolomic, geographic, and seasonal analysis of the contribution of pollen-derived adenosine to allergic sensitization.

Background: Studies on ragweed and birch pollen extracts suggested that the adenosine content is an important factor in allergic sensitization. However, exposure levels from other pollens and considerations of geographic and seasonal factors have not been evaluated.

Objective: This study compared the metabolite profile of pollen species important for allergic disease, specifically measured the adenosine content, and evaluated exposure to pollen-derived adenosine.

A Structural Basis for Biguanide Activity.

Metformin is the most commonly prescribed treatment for type II diabetes and related disorders; however, molecular insights into its mode(s) of action have been limited by an absence of structural data. Structural considerations along with a growing body of literature demonstrating its effects on one-carbon metabolism suggest the possibility of folate mimicry and anti-folate activity. Motivated by the growing recognition that anti-diabetic biguanides may act directly upon the gut microbiome, we have determined structures of the complexes formed between the anti-diabetic biguanides (phenformin, buformin, and metformin) and Escherichia coli dihydrofolate reductase (ecDHFR) based on nuclear magnetic resonance, crystallographic, and molecular modeling studies.

APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress.

The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain.

Comparison of fipronil sources in North Carolina surface water and identification of a novel fipronil transformation product in recycled wastewater.

Fipronil is a phenylpyrazole insecticide that is widely used in residential and agricultural settings to control ants, roaches, termites, and other pests. Fipronil and its transformation products have been found in a variety of environmental matrices, but the source[s] which makes the greatest contribution to fipronil in surface water has yet to be determined. A sampling effort designed to prioritize known fipronil inputs (golf courses, residential areas, biosolids application sites and wastewater facilities) was conducted in North Carolina to learn more about the origins of fipronil in surface water.

Unfolding the HIV-1 reverse transcriptase RNase H domain--how to lose a molecular tug-of-war.

Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit.

Asymmetric conformational maturation of HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase utilizes a metamorphic polymerase domain that is able to adopt two alternate structures that fulfill catalytic and structural roles, thereby minimizing its coding requirements. This ambiguity introduces folding challenges that are met by a complex maturation process. We have investigated this conformational maturation using NMR studies of methyl-labeled RT for the slower processes in combination with molecular dynamics simulations for rapid processes.

Characterization of the redox transition of the XRCC1 N-terminal domain.

XRCC1, a scaffold protein involved in DNA repair, contains an N-terminal domain (X1NTD) that interacts specifically with DNA polymerase β. It was recently discovered that X1NTD contains a disulfide switch that allows it to adopt either of two metamorphic structures. In the present study, we demonstrate that formation of an N-terminal proline carbimate adduct resulting from the nonenzymatic reaction of Pro2 with CO2 is essential for stabilizing the oxidized structure, X1NTDox.

Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation.

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding.