A Protein Misfolding Shaking Amplification-based method for the spontaneous generation of hundreds of bona fide prions.

Prion diseases are a group of rapidly progressing neurodegenerative disorders caused by the misfolding of the endogenous prion protein (PrPC) into a pathogenic form (PrPSc). This process, despite being the central event underlying these disorders, remains largely unknown at a molecular level, precluding the prediction of new potential outbreaks or interspecies transmission incidents. In this work, we present a method to generate bona fide recombinant prions de novo, allowing a comprehensive analysis of protein misfolding across a wide range of prion proteins from mammalian species. We study more than 380 different prion proteins from mammals and classify them according to their spontaneous misfolding propensity and their conformational variability. This study aims to address fundamental questions in the prion research field such as defining infectivity determinants, interspecies transmission barriers or the structural influence of specific amino acids and provide invaluable information for future diagnosis and therapy applications.

Benzylic Radical Stabilization Permits Ether Formation During Darobactin Biosynthesis.

The Gram-negative selective antibiotic darobactin A has attracted interest owing to its intriguing fused bicyclic structure and unique mode of action. Biosynthetic studies have revealed that darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP). During maturation, the darobactin precursor peptide (DarA) is modified by a radical S-adenosyl methionine (rSAM)-dependent enzyme (DarE) to contain ether and C-C crosslinks. In this work, we describe the enzymatic tolerance of DarE using a panel of DarA variants, revealing that DarE can install the ether and C-C crosslinks independently and in different locations on DarA. These efforts produced 57 darobactin variants, 50 of which were enzymatically modified. Several new variants with fused bicyclic structures were characterized, including darobactin W3Y, which replaces tryptophan with tyrosine at the twice-modified central position, and darobactin K5F, which displays a fused diether ring pattern. Three additional darobactin variants contained fused diether macrocycles, leading us to investigate the origin of ether versus C-C crosslink formation. Computational analyses found that more stable and long-lived Cß radicals found on aromatic amino acids correlated with ether formation. Further, molecular docking and calculated transition state structures provide support for the different indole connectivity observed for ether (Trp-C7) and C-C (Trp-C6) crosslink formation. We also provide experimental evidence for a ß-oxotryptophan modification, a proposed intermediate during ether crosslink formation. Finally, mutational analysis of the DarA leader region and protein structural predictions identified which residues were dispensable for processing and others that govern substrate engagement by DarE. Our work informs on darobactin scaffold engineering and sheds additional light on the underlying principles of rSAM catalysis.

The catalytic role of glutathione transferases in heterologous anthocyanin biosynthesis.

Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker's yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants' heterologous microbial production and could impact the breeding of industrial and ornamental plants.

The NCIWEB Server: A Novel Implementation of the Noncovalent Interactions Index for Biomolecular Systems.

It is well-known that the activity and function of proteins is strictly correlated with their secondary, tertiary, and quaternary structures. Their biological role is regulated by their conformational flexibility and global fold, which, in turn, is largely governed by complex noncovalent interaction networks. Because of the large size of proteins, the analysis of their noncovalent interaction networks is challenging, but can provide insights into the energetics of conformational changes or protein-protein and protein-ligand interactions. The noncovalent interaction (NCI) index, based on the reduced density gradient, is a well-established tool for the detection of weak contacts in biological systems. In this work, we present a web-based application to expand the use of this index to proteins, which only requires a molecular structure as input and provides a mapping of the number, type, and strength of noncovalent interactions. Structure preparation is automated and allows direct importing from the PDB database, making this server (https://nciweb.dsi.upmc.fr) accessible to scientists with limited experience in bioinformatics. A quick overview of this tool and concise instructions are presented, together with an illustrative application.

Amyloid Fibrils Formed by Short Prion-Inspired Peptides Are Metalloenzymes.

Enzymes typically fold into defined 3D protein structures exhibiting a high catalytic efficiency and selectivity. It has been proposed that the earliest enzymes may have arisen from the self-assembly of short peptides into supramolecular amyloid-like structures. Several artificial amyloids have been shown to display catalytic activity while offering advantages over natural enzymes in terms of modularity, flexibility, stability, and reusability. Hydrolases, especially esterases, are the most common artificial amyloid-like nanozymes with some reported to act as carbonic anhydrases (CA). Their hydrolytic activity is often dependent on the binding of metallic cofactors through a coordination triad composed of His residues in the ß-strands, which mimic the arrangement found in natural metalloenzymes. Tyr residues contribute to the coordination of metal ions in the active center of metalloproteins; however, their use has been mostly neglected in the design of metal-containing amyloid-based nanozymes. We recently reported that four different polar prion-inspired heptapeptides spontaneously self-assembled into amyloid fibrils. Their sequences lack His but contain three alternate Tyr residues exposed to solvent. We combine experiments and simulations to demonstrate that the amyloid fibrils formed by these peptides can efficiently coordinate and retain different divalent metal cations, functioning as both metal scavengers and nanozymes. The metallized fibrils exhibit esterase and CA activities without the need for a histidine triad. These findings highlight the functional versatility of prion-inspired peptide assemblies and provide a new sequential context for the creation of artificial metalloenzymes. Furthermore, our data support amyloid-like structures acting as ancestral catalysts at the origin of life.

Unraveling the Photophysical Characteristics, Aromaticity, and Stability of π-Extended Acene-Quinodimethyl Thioamides†.

Poly-aromatic systems that contain quinodimethyl (QDM) units are appealing for several photonic and spintronic applications owing to the unique electronic structure, aromaticity, and spin state(s) of the QDM ring. Herein, we report the synthesis and characterization of novel QDM-based chromophores 1-3, which exhibit unique photo-excited behavior and aromaticity. Extending the aromatic core with a biphenyl/phenanthryl- and a pyrrolo-fragment led to reducing the optoelectronic bandgap and modulating the photophysics QDM 1-3. Yet, QDM 2 and 3 suffer from "aromaticity imbalance" and become relatively unstable compared to the parent compound QDM 1. Further assessment of local aromaticity using computational tools revealed that the pseudo-quinoidal ring B is the main driving force allowing to easily populate the excited triplet state of these chromophores. The present study provides complementary guidelines for designing novel non-classical poly-aromatic systems.

Molecular Recognition of Glycan-Bearing Glycomacromolecules Presented at Membrane Surfaces by Lectins: An NMR View.

Lectin-glycan interactions are at the heart of a multitude of biological events. Glycans are usually presented in a multivalent manner on the cell surface as part of the so-called glycocalyx, where they interact with other entities. This multivalent presentation allows us to overcome the typical low affinities found for individual glycan-lectin interactions. Indeed, the presentation of glycans may drastically impact their binding by lectins, highly affecting the corresponding binding affinity and even selectivity. In this context, we herein present the study of the interaction of a variety of homo- and heteromultivalent lactose-functionalized glycomacromolecules and their lipid conjugates with two human galectins. We have employed as ligands the glycomacromolecules, as well as liposomes decorated with those structures, to evaluate their interactions in a cell-mimicking environment. Key details of the interaction have been unravelled by NMR experiments, both from the ligand and receptor perspectives, complemented by cryo-electron microscopy methods and molecular dynamics simulations.

Accurate Prediction of Enzyme Thermostabilization with Rosetta Using AlphaFold Ensembles.

Thermostability enhancement is a fundamental aspect of protein engineering as a biocatalyst's half-life is key for its industrial and biotechnological application, particularly at high temperatures and under harsh conditions. Thermostability changes upon mutation originate from modifications of the free energy of unfolding (ΔGu), making thermostabilization extremely challenging to predict with computational methods. In this contribution, we combine global conformational sampling with energy prediction using AlphaFold and Rosetta to develop a new computational protocol for the quantitative prediction of thermostability changes upon laboratory evolution of acyltransferase LovD and lipase LipA. We highlight how using an ensemble of protein conformations rather than a single three-dimensional model is mandatory for accurate thermostability predictions. By comparing our approaches with existing ones, we show that ensembles based on AlphaFold models provide more accurate and robust calculated thermostability trends than ensembles based solely on crystallographic structures as the latter introduce a strong distortion (scaffold bias) in computed thermostabilities. Eliminating this bias is critical for computer-guided enzyme design and evaluating the effect of multiple mutations on protein stability.

Iridium-Catalyzed ortho-Selective Borylation of Aromatic Amides Enabled by 5-Trifluoromethylated Bipyridine Ligands.

Iridium-catalyzed borylations of aromatic C-H bonds are highly attractive transformations because of the diversification possibilities offered by the resulting boronates. These transformations are best carried out using bidentate bipyridine or phenanthroline ligands, and tend to be governed by steric factors, therefore resulting in the competitive functionalization of meta and/or para positions. We have now discovered that a subtle change in the bipyridine ligand, namely, the introduction of a CF3 substituent at position 5, enables a complete change of regioselectivity in the borylation of aromatic amides, allowing the synthesis of a wide variety of ortho-borylated derivatives. Importantly, thorough computational studies suggest that the exquisite regio- and chemoselectivity stems from unusual outer-sphere interactions between the amide group of the substrate and the CF3 -substituted aryl ring of the bipyridine ligand.

Conformationally Restricted ß-Sheet Breaker Peptides Incorporating Cyclic α-Methylisoserine Sulfamidates.

Peptides containing variations of the ß-amyloid hydrophobic core and five-membered sulfamidates derived from ß-amino acid α-methylisoserine have been synthesized and fully characterized in the gas phase, solid state and in aqueous solution by a combination of experimental and computational techniques. The cyclic sulfamidate group effectively locks the secondary structure at the N-terminus of such hybrid peptides imposing a conformational restriction and stabilizing non-extended structures. This conformational bias, which is maintained in the gas phase, solid state and aqueous solution, is shown to be resistant to structure templating through assays of in vitro ß-amyloid aggregation, acting as ß-sheet breaker peptides with moderate activity.

Regioselective Synthesis and Molecular Docking Studies of 1,5-Disubstituted 1,2,3-Triazole Derivatives of Pyrimidine Nucleobases.

1,2,3-triazoles are versatile building blocks with growing interest in medicinal chemistry. For this reason, organic chemistry focuses on the development of new synthetic pathways to obtain 1,2,3-triazole derivatives, especially with pyridine moieties. In this work, a novel series of 1,5-disubstituted-1,2,3-triazoles functionalized with pyrimidine nucleobases were prepared via 1,3-dipolar cycloaddition reaction in a regioselective manner for the first time. The N1-propargyl nucleobases, used as an alkyne intermediate, were obtained in high yields (87-92%) with a new two-step procedure that selectively led to the monoalkylated compounds. Then, FeCl3 was employed as an efficient Lewis acid catalyst for 1,3-dipolar cycloaddition between different aryl and benzyl azides and the N1-propargyl nucleobases previously synthesized. This new protocol allows the synthesis of a series of new 1,2,3-triazole derivatives with good to excellent yields (82-92%). The ADME (Absorption, Distribution, Metabolism, and Excretion) analysis showed good pharmacokinetic properties and no violations of Lipinsky's rules, suggesting an appropriate drug likeness for these new compounds. Molecular docking simulations, conducted on different targets, revealed that two of these new hybrids could be potential ligands for viral and bacterial protein receptors such as human norovirus capsid protein, SARS-CoV-2 NSP13 helicase, and metallo-ß-lactamase.

Immobilization of Biantennary N-Glycans Leads to Branch Specific Epitope Recognition by LSECtin.

The molecular recognition features of LSECtin toward asymmetric N-glycans have been scrutinized by NMR and compared to those occurring in glycan microarrays. A pair of positional glycan isomers (LDN3 and LDN6), a nonelongated GlcNAc4Man3 N-glycan (G0), and the minimum binding epitope (the GlcNAcß1-2Man disaccharide) have been used to shed light on the preferred binding modes under both experimental conditions. Strikingly, both asymmetric LDN3 and LDN6 N-glycans are recognized by LSECtin with similar affinities in solution, in sharp contrast to the results obtained when those glycans are presented on microarrays, where only LDN6 was efficiently recognized by the lectin. Thus, different results can be obtained using different experimental approaches, pointing out the tremendous difficulty of translating in vitro results to the in vivo environment.

Development of a Highly Potent Transthyretin Amyloidogenesis Inhibitor: Design, Synthesis, and Evaluation.

Transthyretin amyloidosis (ATTR) is a group of fatal diseases described by the misfolding and amyloid deposition of transthyretin (TTR). Discovering small molecules that bind and stabilize the TTR tetramer, preventing its dissociation and subsequent aggregation, is a therapeutic strategy for these pathologies. Departing from the crystal structure of TTR in complex with tolcapone, a potent binder in clinical trials for ATTR, we combined rational design and molecular dynamics (MD) simulations to generate a series of novel halogenated kinetic stabilizers. Among them, M-23 displays one of the highest affinities for TTR described so far. The TTR/M-23 crystal structure confirmed the formation of unprecedented protein-ligand contacts, as predicted by MD simulations, leading to an enhanced tetramer stability both in vitro and in whole serum. We demonstrate that MD-assisted design of TTR ligands constitutes a new avenue for discovering molecules that, like M-23, hold the potential to become highly potent drugs to treat ATTR.

Assessing the Mobility of Severe Acute Respiratory Syndrome Coronavirus-2 Spike Protein Glycans by Structural and Computational Methods.

Two years after its emergence, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several vaccines. The extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin converting enzyme 2 (ACE2) through its receptor binding domain (RBD), is the major target of neutralizing antibodies. Like to many other viral fusion proteins, the SARS-CoV-2 spike protein utilizes a glycan shield to thwart the host immune response. To grasp the influence of chemical signatures on carbohydrate mobility and reconcile the cryo-EM density of specific glycans we combined our cryo-EM map of the S ectodomain to 4.1 Å resolution, reconstructed from a limited number of particles, and all-atom molecular dynamics simulations. Chemical modifications modeled on representative glycans (defucosylation, sialylation and addition of terminal LacNAc units) show no significant influence on either protein shielding or glycan flexibility. By estimating at selected sites the local correlation between the full density map and atomic model-based maps derived from molecular dynamics simulations, we provide insight into the geometries of the α-Man-(1→3)-[α-Man-(1→6)-]-ß-Man-(1→4)-ß-GlcNAc(1→4)-ß-GlcNAc core common to all N-glycosylation sites.

A Computational Perspective on Molecular Recognition by Galectins.

This article presents an overview of recent computational studies dedicated to the analysis of binding between galectins and small-molecule ligands. We first present a summary of the most popular simulation techniques adopted for calculating binding poses and binding energies and then discuss relevant examples reported in the literature for the three main classes of galectins (dimeric, tandem, and chimera). We show that simulation of galectin-ligand interactions is a mature field that has proven invaluable for completing and unraveling experimental observations. Future perspectives to further improve the accuracy and cost-effectiveness of existing computational approaches will involve the development of new schemes to account for solvation and entropy effects, which represent the main current limitations to the accuracy of computational results.

Cluster Assembled Silicon-Lithium Nanostructures: A Nanowire Confined Inside a Carbon Nanotube.

We computationally explore an alternative to stabilize one-dimensional (1D) silicon-lithium nanowires (NWs). The Li12Si9 Zintl phase exhibits the NW [ Li 6 Si 5 ] ∞ 1 , combined with Y-shaped Si4 structures. Interestingly, this NW could be assembled from the stacking of the Li6Si5 aromatic cluster. The [ Li 6 Si 5 ] ∞ 1 @CNT nanocomposite has been investigated with density functional theory (DFT), including molecular dynamics simulations and electronic structure calculations. We found that van der Waals interaction between Li's and CNT's walls is relevant for stabilizing this hybrid nanocomposite. This work suggests that nanostructured confinement (within CNTs) may be an alternative to stabilize this free NW, cleaning its properties regarding Li12Si9 solid phase, i.e., metallic character, concerning the perturbation provided by their environment in the Li12Si7 compound.

Bacteriophage PRD1 as a nanoscaffold for drug loading.

Viruses are very attractive biomaterials owing to their capability as nanocarriers of genetic material. Efforts have been made to functionalize self-assembling viral protein capsids on their exterior or interior to selectively take up different payloads. PRD1 is a double-stranded DNA bacteriophage comprising an icosahedral protein outer capsid and an inner lipidic vesicle. Here, we report the three-dimensional structure of PRD1 in complex with the antipsychotic drug chlorpromazine (CPZ) by cryo-electron microscopy. We show that the jellyrolls of the viral major capsid protein P3, protruding outwards from the capsid shell, serve as scaffolds for loading heterocyclic CPZ molecules. Additional X-ray studies and molecular dynamics simulations show the binding modes and organization of CPZ molecules when complexed with P3 only and onto the virion surface. Collectively, we provide a proof of concept for the possible use of the lattice-like organisation and the quasi-symmetric morphology of virus capsomers for loading heterocyclic drugs with defined properties.

Atomistic insights into the structure of heptapeptide nanofibers.

Artificial amyloid-like nanofibers formed from short peptides are emerging as new supramolecular structures for catalysis and advanced materials. In this work, we analyze, by means of computational approaches, the preferred atomistic fibrillar architectures that result from the self-assembly of polar NY7, NF7, SY7, SF7, and GY7 peptides into steric zippers formed by two ß-sheets (describing an individual steric zipper) and by four ß-sheets. For all heptapeptides, except GY7, parallel ß-sheet organizations with polar residues packed at the steric zipper appear to be the preferred assemblies for the two ß-sheets system due to the formation of a strong network of hydrogen bonds. For GY7, however, an antiparallel organization with glycine at the steric zipper is the most stable one. The preferred architecture is mostly conserved when enlarging our model from two to four ß-sheets. The present work shows that the relative stability of different architectures results from a delicate balance between peptide composition, side chain hydrophobicity, and non-covalent interactions at the interface and provides the basis for a rational design of new improved artificial prion-inspired materials.

Impact of Cu(II) and Al(III) on the conformational landscape of amyloidß1-42.

Metal ions have been found to play an important role in the formation of extracellular ß-amyloid plaques, a major hallmark of Alzheimer's disease. In the present study, the conformational landscape of Aß42 with Al(iii) and Cu(ii) has been explored using Gaussian accelerated molecular dynamics. Both metals reduce the flexibility of the peptide and entail a higher structural organization, although to different degrees. As a general trend, Cu(ii) binding leads to an increased α-helix content and to the formation of two α-helices that tend to organize in a U-shape. By contrast, most Al(iii) complexes induce a decrease in helical content, leading to more extended structures that favor the appearance of transitory ß-strands.

Enthalpy-Entropy Compensation in Biomolecular Recognition: A Computational Perspective.

This mini-review provides an overview of the enthalpy-entropy compensation phenomenon in the simulation of biomacromolecular recognition, with particular emphasis on ligand binding. We approach this complex phenomenon from the point of view of practical computational chemistry. Without providing a detailed description of the plethora of existing methodologies already reviewed in depth elsewhere, we present a series of examples to illustrate different approaches to interpret and predict compensation phenomena at an atomistic level, which is far from trivial to predict using canonical, classic textbook assumptions.