Adenosine nucleotide binding sites on Complex V from diverse organisms

Using in silico docking approaches, we scan the various subunits of Complex V (FoF1ATPase) for putative adenosine nucleotide binding sites. We find that multiple generic ADP/ATP binding sites are present on the alpha-beta binding sites and a conserved ATP binding site is present on the epsilon subunit. These findings support the murburn model of Complex V.

Bioinformatic-Based Approaches for Disease-Resistance Gene Discovery in Plants

Pathogens are among the most limiting factors for crop success and expansion. Thus, finding the underlying genetic cause of pathogen resistance is the main goal for plant geneticists. The activation of a plant’s immune system is mediated by the presence of specific receptors known as disease-resistance genes (R genes). Typical R genes encode functional immune receptors with nucleotide-binding sites (NBS) and leucine-rich repeat (LRR) domains, making the NBS-LRRs the largest family of plant resistance genes. Establishing host resistance is crucial for plant growth and crop yield but also for reducing pesticide use. In this regard, pyramiding R genes is thought to be the most ecologically friendly way to enhance the durability of resistance. To accomplish this, researchers must first identify the related genes, or linked markers, within the genomes. However, the duplicated nature, with the presence of frequent paralogues, and clustered characteristic of NLRs make them difficult to predict with the classic automatic gene annotation pipelines. In the last several years, efforts have been made to develop new methods leading to a proliferation of reports on cloned genes. Herein, we review the bioinformatic tools to assist the discovery of R genes in plants, focusing on well-established pipelines with an important computer-based component.

Energy transfer between the nicotinamide nucleotide transhydrogenase and ATP synthase of Escherichia coli

Membrane bound nicotinamide nucleotide transhydrogenase (TH) catalyses the hydride transfer from NADH to NADP+. Under physiological conditions, this reaction is endergonic and must be energized by the pmf, coupled to transmembrane proton transport. Recent structures of transhydrogenase holoenzymes suggest new mechanistic details, how the long-distance coupling between hydride transfer in the peripheral nucleotide binding sites and the membrane-localized proton transfer occurs that now must be tested experimentally. Here, we provide protocols for the efficient expression and purification of the Escherichia coli transhydrogenase and its reconstitution into liposomes, alone or together with the Escherichia coli F1F0 ATP synthase. We show that E. coli transhydrogenase is a reversible enzyme that can also work as a NADPH-driven proton pump. In liposomes containing both enzymes, NADPH driven H+-transport by TH is sufficient to instantly fuel ATP synthesis, which adds TH to the pool of pmf generating enzymes. If the same liposomes are energized with ATP, NADPH production by TH is stimulated > sixfold both by a pH gradient or a membrane potential. The presented protocols and results reinforce the tight coupling between hydride transfer in the peripheral nucleotide binding sites and transmembrane proton transport and provide powerful tools to investigate their coupling mechanism.

Molecular basis of cholesterol efflux via ABCG subfamily transporters

The ABCG1 homodimer (G1) and ABCG5–ABCG8 heterodimer (G5G8), two members of the adenosine triphosphate (ATP)–binding cassette (ABC) transporter G family, are required for maintenance of cellular cholesterol levels. G5G8 mediates secretion of neutral sterols into bile and the gut lumen, whereas G1 transports cholesterol from macrophages to high-density lipoproteins (HDLs). The mechanisms used by G5G8 and G1 to recognize and export sterols remain unclear. Here, we report cryoelectron microscopy (cryo-EM) structures of human G5G8 in sterol-bound and human G1 in cholesterol- and ATP-bound states. Both transporters have a sterol-binding site that is accessible from the cytosolic leaflet. A second site is present midway through the transmembrane domains of G5G8. The Walker A motif of G8 adopts a unique conformation that accounts for the marked asymmetry in ATPase activities between the two nucleotide-binding sites of G5G8. These structures, along with functional validation studies, provide a mechanistic framework for understanding cholesterol efflux via ABC transporters.

Structural and Functional Genomics of the Resistance of Cacao to Phytophthora palmivora

Black pod disease, caused by Phytophthora spp., is one of the main diseases that attack cocoa plantations. This study validated, by association mapping, 29 SSR molecular markers flanking to QTL (Quantitative Trait Loci) associated with Phytophthora palmivora Butler (Butler) (PP) resistance, in three local ancient varieties of the Bahia (Comum, Pará, and Maranhão), varieties that have a high potential in the production of gourmet chocolate. Four SSR loci associated with resistance to PP were detected, two on chromosome 8, explaining 7.43% and 3.72% of the Phenotypic Variation (%PV), one on chromosome 2 explaining 2.71%PV and one on chromosome 3 explaining 1.93%PV. A functional domains-based annotation was carried out, in two Theobroma cacao (CRIOLLO and MATINA) reference genomes, of 20 QTL regions associated with cocoa resistance to the pathogen. It was identified 164 (genome CRIOLLO) and 160 (genome MATINA) candidate genes, hypothetically involved in the recognition and activation of responses in the interaction with the pathogen. Genomic regions rich in genes with Coiled-coils (CC), nucleotide binding sites (NBS) and Leucine-rich repeat (LRR) domains were identified on chromosomes 1, 3, 6, 8, and 10, likewise, regions rich in Receptor-like Kinase domain (RLK) and Ginkbilobin2 (GNK2) domains were identified in chromosomes 4 and 6.

Long-range allosteric effects - How adenine nucleotides shape the conformational landscape of the Ski2-like RNA helicase, Brr2

ABSTRACTBrr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar, but enzymatically inactive C-terminal helicase cassette. Both cassettes contain a nucleotide binding pocket. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and how occupancy of the nucleotide binding sites may influence each other. Our results show that Brr2 exhibits high specificity for adenine nucleotides with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active N-terminal cassette, largely determined by slow nucleotide release. Mutations at the inter-cassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette, 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate the long-range allosteric effects. Together, our results reveal intricate networks of intra-molecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited for regulating the enzyme during splicing.

HIV Pre-Exposure Prophylaxis Adherence Test Using Reverse Transcription Isothermal Amplification Inhibition Assay

Current HIV antiretroviral (ART) or pre-exposure prophylaxis (PrEP) therapy adherence monitoring relies on either patient self-reported adherence or monitoring drug dispensing, which are not reliable. We propose an objective adherence monitoring assay which directly measures nucleotide reverse transcriptase inhibitor (NRTI) concentration using a reverse transcription isothermal amplification inhibition assay. We measure the concentration of Tenofovir diphosphate (TFV-DP), a deoxyadenosine triphosphate (dATP) analog and an active NRTI compound and long-term adherence marker for PrEP, by measuring the inhibition of the reverse transcription of an RNA template. The completion or inhibition of reverse transcription is evaluated by Recombinase Polymerase Amplification (RPA), an isothermal nucleic acid amplification assay. We present and validate a model that predicts the amplification probability as function of dATP and TFV-DP concentrations, nucleotide binding sites on the RNA template, and the RNA template concentration. The model helps to rationally design and optimize the assay to operate at clinically relevant TFV-DP concentrations. We provide statistical analysis that demonstrates how the assay can be used as a qualitative or semi-quantitative tool for measuring adherence to NRTI drugs and used to support patient compliance.

Nucleotide-binding sites can enhance N-acylation of nearby protein lysine residues

Acyl-CoAs are reactive metabolites that can non-enzymatically S-acylate and N-acylate protein cysteine and lysine residues, respectively. N-acylation is irreversible and enhanced if a nearby cysteine residue undergoes an initial reversible S-acylation, as proximity leads to rapid S → N-transfer of the acyl moiety. We reasoned that protein-bound acyl-CoA could also facilitate S → N-transfer of acyl groups to proximal lysine residues. Furthermore, as CoA contains an ADP backbone this may extend beyond CoA-binding sites and include abundant Rossmann-fold motifs that bind the ADP moiety of NADH, NADPH, FADH and ATP. Here, we show that excess nucleotides decrease protein lysine N-acetylation in vitro. Furthermore, by generating modelled structures of proteins N-acetylated in mouse liver, we show that proximity to a nucleotide-binding site increases the risk of N-acetylation and identify where nucleotide binding could enhance N-acylation in vivo. Finally, using glutamate dehydrogenase as a case study, we observe increased in vitro lysine N-malonylation by malonyl-CoA near nucleotide-binding sites which overlaps with in vivo N-acetylation and N-succinylation. Furthermore, excess NADPH, GTP and ADP greatly diminish N-malonylation near their nucleotide-binding sites, but not at distant lysine residues. Thus, lysine N-acylation by acyl-CoAs is enhanced by nucleotide-binding sites and may contribute to higher stoichiometry protein N-acylation in vivo.

Mapping Powdery Mildew (Blumeria graminis f. sp. tritici) Resistance in Wild and Cultivated Tetraploid Wheats

Wheat is the most widely grown crop and represents the staple food for one third of the world’s population. Wheat is attacked by a large variety of pathogens and the use of resistant cultivars is an effective and environmentally safe strategy for controlling diseases and eliminating the use of fungicides. In this study, a collection of wild and cultivated tetraploid wheats (Triticum turgidum) were evaluated for seedling resistance (SR) and adult plant resistance (APR) to powdery mildew (Blumeria graminis) and genotyped with a 90K single nucleotide polymorphism (SNP) array to identify new sources of resistance genes. The genome-wide association mapping detected 18 quantitative trait loci (QTL) for APR and 8 QTL for SR, four of which were identical or at least closely linked to four QTL for APR. Thirteen candidate genes, containing nucleotide binding sites and leucine-rich repeats, were localized in the confidence intervals of the QTL-tagging SNPs. The marker IWB6155, associated to QPm.mgb-1AS, was located within the gene TRITD1Av1G004560 coding for a disease resistance protein. While most of the identified QTL were described previously, five QTL for APR (QPm.mgb-1AS, QPm.mgb-2BS, QPm.mgb-3BL.1, QPm.mgb-4BL, QPm.mgb-7BS.1) and three QTL for SR (QPm.mgb-3BL.3, QPm.mgb-5AL.2, QPm.mgb-7BS.2) were mapped on chromosome regions where no resistance gene was reported before. The novel QTL/genes can contribute to enriching the resistance sources available to breeders.

High-resolution structures of the SARS-CoV-2 2′-O-methyltransferase reveal strategies for structure-based inhibitor design

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2′-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.