An Alternative Binding Mode of IGHV3-53 Antibodies to the SARS-CoV-2 Receptor Binding Domain
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59 BASIC BIOLOGICAL SCIENCES↗
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Salmonella Typhoid Toxin PltB Subunit and Its Non-typhoidal Salmonella Ortholog Confer Differential Host Adaptation and Virulence
Typhoidal and non-typhoidal Salmonelleae (NTS) cause typhoid fever and gastroenteritis, respectively, in humans. Salmonella typhoid toxin contributes to typhoid disease progression and chronic infection, but little is known about the role of its NTS ortholog. We found that typhoid toxin and its NTS ortholog induce different clinical presentations. The PltB subunit of each toxin exhibits different glycan-binding preferences that correlate with glycan expression profiles of host cells targeted by each bacterium at the primary infection or intoxication sites. Through co-crystal structures of PltB subunits bound to specific glycan receptor moieties, we show that they induce markedly different glycan-binding preferences and virulence outcomes. Furthermore, immunization with the NTS S. Javiana or its toxin offers cross-reactive protection against lethal-dose typhoid toxin challenge. Cumulatively, these results offer insights into the evolution of host adaptations in Salmonella AB toxins, their cell and tissue tropisms, and the design for improved typhoid vaccines and therapeutics.
59 BASIC BIOLOGICAL SCIENCES↗
Combining Force Fields and Neural Networks for an Accurate Representation of Chemically Diverse Molecular Interactions
A key goal of molecular modeling is the accurate reproduction of the true quantum mechanical potential energy of arbitrary molecular ensembles with a tractable classical approximation. The challenges are that analytical expressions found in general purpose force fields struggle to faithfully represent the intermolecular quantum potential energy surface at close distances and in strong interaction regimes; that the more accurate neural network approximations do not capture crucial physics concepts, e.g., nonadditive inductive contributions and application of electric fields; and that the ultra-accurate narrowly targeted models have difficulty generalizing to the entire chemical space. We therefore designed a hybrid wide-coverage intermolecular interaction model consisting of an analytically polarizable force field combined with a short-range neural network correction for the total intermolecular interaction energy. Here, we describe the methodology and apply the model to accurately determine the properties of water, the free energy of solvation of neutral and charged molecules, and the binding free energy of ligands to proteins. The correction is subtyped for distinct chemical species to match the underlying force field, to segment and reduce the amount of quantum training data, and to increase accuracy and computational speed. For the systems considered, the hybrid ab initio parametrized Hamiltonian reproduces the two-body dimer quantum mechanics (QM) energies to within 0.03 kcal/mol and the nonadditive many-molecule contributions to within 2%. Simulations of molecular systems using this interaction model run at speeds of several nanoseconds per day.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence
Abstract cGAS is an evolutionarily conserved enzyme that has a pivotal role in immune defence against infection 1–3 . In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP–AMP (cGAMP) 4,5 , which leads to the expression of antimicrobial genes 6,7 . In bacteria, cyclic dinucleotide (CDN)-based anti-phage signalling systems (CBASS) have been discovered 8–11 . These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria on phage infection, thereby stopping phage spread. Of the CBASS systems reported, approximately 39% contain Cap2 and Cap3 , which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating enzymes, respectively 8,12 . Although these proteins are required to prevent infection of some bacteriophages 8 , the mechanism by which the enzymatic activities exert an anti-phage effect is unknown. Here we show that Cap2 forms a thioester bond with the C-terminal glycine of cGAS and promotes conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. The covalent conjugation of cGAS increases the production of cGAMP. Using a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signalling by binding tightly to cGAMP (dissociation constant of approximately 30 nM) and sequestering it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that was bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrates an arms race between bacteria and viruses through controlling CDN levels.
Science & Technology - Other Topics↗
Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations
Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.
60 APPLIED LIFE SCIENCES↗
NTRC regulates CP12 to activate Calvin–Benson cycle during cold acclimation
NADPH-dependent thioredoxin reductase C (NTRC) is a chloroplast redox regulator in algae and plants. Here, we used site-specific mutation analyses of the thioredoxin domain active site of NTRC in the green alga Chlamydomonas reinhardtii to show that NTRC mediates cold tolerance in a redox-dependent manner. By means of coimmunoprecipitation and mass spectrometry, a redox- and cold-dependent binding of the Calvin–Benson Cycle Protein 12 (CP12) to NTRC was identified. NTRC was subsequently demonstrated to directly reduce CP12 of C. reinhardtii as well as that of the vascular plant Arabidopsis thaliana in vitro. As a scaffold protein, CP12 joins the Calvin–Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to form an autoinhibitory supracomplex. Using size-exclusion chromatography, NTRC from both organisms was shown to control the integrity of this complex in vitro and thereby PRK and GAPDH activities in the cold. Thus, NTRC apparently reduces CP12, hence triggering the dissociation of the PRK/CP12/GAPDH complex in the cold. Like the ntrc::aphVIII mutant, CRISPR-based cp12::emx1 mutants also exhibited a redox-dependent cold phenotype. In addition, CP12 deletion resulted in robust decreases in both PRK and GAPDH protein levels implying a protein protection effect of CP12. Both CP12 functions are critical for preparing a repertoire of enzymes for rapid activation in response to environmental changes. This provides a crucial mechanism for cold acclimation.
54 ENVIRONMENTAL SCIENCES↗
Discovery of GuaB inhibitors with efficacy against Acinetobacter baumannii infection
ABSTRACT Guanine nucleotides are required for growth and viability of cells due to their structural role in DNA and RNA, and their regulatory roles in translation, signal transduction, and cell division. The natural antibiotic mycophenolic acid (MPA) targets the rate-limiting step inde novoguanine nucleotide biosynthesis executed by inosine-5´-monophosphate dehydrogenase (IMPDH). MPA is used clinically as an immunosuppressant, but whetherin vivoinhibition of bacterial IMPDH (GuaB) is a valid antibacterial strategy is controversial. Here, we describe the discovery of extremely potent small molecule GuaB inhibitors (GuaBi) specific to pathogenic bacteria with a low frequency of on-target spontaneous resistance and bactericidal efficacyin vivoagainstAcinetobacter baumanniimouse models of infection. The spectrum of GuaBi activity includes multidrug-resistant pathogens that are a critical priority of new antibiotic development. Co-crystal structures ofA. baumannii, Staphylococcus aureus, andEscherichia coliGuaB proteins bound to inhibitors show comparable binding modes of GuaBi across species and identifies key binding site residues that are predictive of whole-cell activity across both Gram-positive and Gram-negative clades of Bacteria. The clearin vivoefficacy of these small molecule GuaB inhibitors in a model ofA. baumanniiinfection validates GuaB as an essential antibiotic target. IMPORTANCE The emergence of multidrug-resistant bacteria worldwide has renewed interest in discovering antibiotics with novel mechanism of action. For the first time ever, we demonstrate that pharmacological inhibition ofde novoguanine biosynthesis is bactericidal in a mouse model ofAcinetobacter baumanniiinfection. Structural analyses of novel inhibitors explain differences in biochemical and whole-cell activity across bacterial clades and underscore why this discovery may have broad translational impact on treatment of the most recalcitrant bacterial infections.
Microbiology↗
Cyanobacteriochromes: A Rainbow of Photoreceptors
Widespread phytochrome photoreceptors use photoisomerization of linear tetrapyrrole (bilin) chromophores to measure the ratio of red to far-red light. Cyanobacteria also contain distantly related cyanobacteriochrome (CBCR) proteins that share the bilin-binding GAF domain of phytochromes but sense other colors of light. CBCR photocycles are extremely diverse, ranging from the near-UV to the near-IR. Photoisomerization of the bilin triggers photoconversion of the CBCR input, thereby modulating the biochemical signaling state of output domains such as histidine kinase bidomains that can interface with cellular signal transduction pathways. CBCRs thus can regulate several aspects of cyanobacterial photobiology, including phototaxis, metabolism of cyclic nucleotide second messengers, and optimization of the cyanobacterial light-harvesting apparatus. This review examines spectral tuning, photoconversion, and photobiology of CBCRs and recent developments in understanding their evolution and in applying them in synthetic biology.
59 BASIC BIOLOGICAL SCIENCES↗
Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action
Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted repeat-lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form two or three intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +8 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea, causal agent of gray mold disease in fruits, vegetables, and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides a structural framework for design of novel, potent antifungal peptides for agricultural use.
59 BASIC BIOLOGICAL SCIENCES↗
Structural and Proteomic Studies of the Aureococcus anophagefferens Virus Demonstrate a Global Distribution of Virus-Encoded Carbohydrate Processing
Viruses modulate the function(s) of environmentally relevant microbial populations, yet considerations of the metabolic capabilities of individual virus particles themselves are rare. We used shotgun proteomics to quantitatively identify 43 virus-encoded proteins packaged within purified Aureococcus anophagefferens Virus (AaV) particles, normalizing data to the per-virion level using a 9.5-Å-resolution molecular reconstruction of the 1900-Å (AaV) particle that we generated with cryogenic electron microscopy. This packaged proteome was used to determine similarities and differences between members of different giant virus families. We noted that proteins involved in sugar degradation and binding (e.g., carbohydrate lyases) were unique to AaV among characterized giant viruses. To determine the extent to which this virally encoded metabolic capability was ecologically relevant, we examined the TARA Oceans dataset and identified genes and transcripts of viral origin. Our analyses demonstrated that putative giant virus carbohydrate lyases represented up to 17% of the marine pool for this function. In total, our observations suggest that the AaV particle has potential prepackaged metabolic capabilities and that these may be found in other giant viruses that are widespread and abundant in global oceans.
59 BASIC BIOLOGICAL SCIENCES↗
Shedding Light on Primary Donors in Photosynthetic Reaction Centers
Chlorophylls (Chl)s exist in a variety of flavors and are ubiquitous in both the energy and electron transfer processes of photosynthesis. The functions they perform often occur on the ultrafast (fs–ns) time scale and until recently, these have been difficult to measure in real time. Further, the complexity of the binding pockets and the resulting protein-matrix effects that alter the respective electronic properties have rendered theoretical modeling of these states difficult. Recent advances in experimental methodology, computational modeling, and emergence of new reaction center (RC) structures have renewed interest in these processes and allowed researchers to elucidate previously ambiguous functions of Chls and related pheophytins. This is complemented by a wealth of experimental data obtained from decades of prior research. Studying the electronic properties of Chl molecules has advanced our understanding of both the nature of the primary charge separation and subsequent electron transfer processes of RCs. In this review, we examine the structures of primary electron donors in Type I and Type II RCs in relation to the vast body of spectroscopic research that has been performed on them to date. Further, we present density functional theory calculations on each oxidized primary donor to study both their electronic properties and our ability to model experimental spectroscopic data. This allows us to directly compare the electronic properties of hetero- and homodimeric RCs.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Targeting a cell surface vitamin D receptor on tumor-associated macrophages in triple-negative breast cancer
Triple-negative breast cancer (TNBC) is an aggressive tumor with limited treatment options and poor prognosis. We applied the in vivo phage display technology to isolate peptides homing to the immunosuppressive cellular microenvironment of TNBC as a strategy for non-malignant target discovery. We identified a cyclic peptide (CSSTRESAC) that specifically binds to a vitamin D receptor, protein disulfide-isomerase A3 (PDIA3) expressed on the cell surface of tumor-associated macrophages (TAM), and targets breast cancer in syngeneic TNBC, non-TNBC xenograft, and transgenic mouse models. Systemic administration of CSSTRESAC to TNBC-bearing mice shifted the cytokine profile toward an antitumor immune response and delayed tumor growth. Moreover, CSSTRESAC enabled ligand-directed theranostic delivery to tumors and a mathematical model confirmed our experimental findings. Finally, in silico analysis showed PDIA3-expressing TAM in TNBC patients. This work uncovers a functional interplay between a cell surface vitamin D receptor in TAM and antitumor immune response that could be therapeutically exploited.
59 BASIC BIOLOGICAL SCIENCES↗
Calcium‐Ion Binding Mediates the Reversible Interconversion of Cis and Trans Peroxido Dicopper Cores
Abstract Coupled dinuclear copper oxygen cores (Cu 2 O 2 ) featured in type III copper proteins (hemocyanin, tyrosinase, catechol oxidase) are vital for O 2 transport and substrate oxidation in many organisms. μ ‐1,2‐ cis peroxido dicopper cores ( C P ) have been proposed as key structures in the early stages of O 2 binding in these proteins; their reversible isomerization to other Cu 2 O 2 cores are directly relevant to enzyme function. Despite the relevance of such species to type III copper proteins and the broader interest in the properties and reactivity of bimetallic C P cores in biological and synthetic systems, the properties and reactivity of C P Cu 2 O 2 species remain largely unexplored. Herein, we report the reversible interconversion of μ ‐1,2‐ trans peroxido ( T P ) and C P dicopper cores. Ca II mediates this process by reversible binding at the Cu 2 O 2 core, highlighting the unique capability for metal‐ion binding events to stabilize novel reactive fragments and control O 2 activation in biomimetic systems.
Vargo, Natasha P.↗
A Structural Perspective on the Alphavirus Life Cycle
Alphaviruses are mosquito-borne, enveloped viruses with a positive-sense, single-stranded RNA genome. Alphaviruses enter host cells via receptor-mediated endocytosis, using various cellular surface receptors such as matrix remodeling-associated protein 8 (MXRA8), low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3), and very low-density lipoprotein receptor (VLDLR), which facilitate binding to the viral glycoproteins. Following entry, viral proteins are expressed and nonstructural proteins assemble into replication complexes in host cells, driving RNA synthesis and genome replication. Viral assembly occurs at the plasma membrane, where nascent virions bud from the host cell in a process driven by capsid and spike proteins. Recent combinatorial structural studies have provided detailed molecular insights into various steps of the alphavirus life cycle. These structural insights into the alphavirus life cycle enhance our understanding of viral replication and assembly, with significant implications for antiviral strategies and the development of alphavirus-based vaccine vectors.
RNA virus↗
Identification of preferred multimodal ligand‐binding regions on IgG1 F C using nuclear magnetic resonance and molecular dynamics simulations
Abstract In this study, the binding of multimodal chromatographic ligands to the IgG1 F C domain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated 15 N‐labeled F C domain indicated that while single‐mode ion exchange ligands interacted very weakly throughout the F C surface, multimodal ligands containing negatively charged and aromatic moieties interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the F C . The multimodal ligand‐binding sites on the F C were concentrated in the hinge region and near the interface of the C H 2 and C H 3 domains. Furthermore, the multimodal binding sites were primarily composed of positively charged, polar, and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on F C corroborated molecular‐level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand‐F C binding in these preferred regions was shown to be electrostatic interactions and π–π stacking of surface‐exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular‐level understanding of multimodal ligand–F C interactions and sets the stage for future analyses of even more complex biotherapeutics.
Gudhka, Ronak B.↗
RNA-guided nucleic acid modifying enzymes and methods of use thereof
The present disclosure provides CasX proteins, nucleic acids encoding the CasX proteins, and modified host cells comprising the CasX proteins and/or nucleic acids encoding same. CasX proteins are useful in a variety of applications, which are provided. The present disclosure provides CasX guide RNAs that bind to and provide sequence specificity to the CasX proteins, nucleic acids encoding the CasX guide RNAs, and modified host cells comprising the CasX guide RNAs and/or nucleic acids encoding same. CasX guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides archaeal Cas9 polypeptides and nucleic acids encoding same, as well as their associated archaeal Cas9 guide RNAs and nucleic acids encoding same.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
RNA-guided nucleic acid modifying enzymes and methods of use thereof
The present disclosure provides CasY proteins, nucleic acids encoding the CasY proteins, and modified host cells comprising the CasY proteins and/or nucleic acids encoding same. CasY proteins are useful in a variety of applications, which are provided. The present disclosure provides CasY guide RNAs that bind to and provide sequence specificity to the CasY proteins, nucleic acids encoding the CasY guide RNAs, and modified host cells comprising the CasY guide RNAs and/or nucleic acids encoding same. CasY guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides methods of identifying a CRISPR RNA-guided endonuclease.
Doudna, Jennifer A.↗
RNA-guided nucleic acid modifying enzymes and methods of use thereof
The present disclosure provides CasX proteins, nucleic acids encoding the CasX proteins, and modified host cells comprising the CasX proteins and/or nucleic acids encoding same. CasX proteins are useful in a variety of applications, which are provided. The present disclosure provides CasX guide RNAs that bind to and provide sequence specificity to the CasX proteins, nucleic acids encoding the CasX guide RNAs, and modified host cells comprising the CasX guide RNAs and/or nucleic acids encoding same. CasX guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides archaeal Cas9 polypeptides and nucleic acids encoding same, as well as their associated archaeal Cas9 guide RNAs and nucleic acids encoding same.
Doudna, Jennifer A.↗