Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.

Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation.

Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts. Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation. Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved, and has important consequences for complex formation and regulation of protein abundance.

Proteome-wide mapping of short-lived proteins in human cells.

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

Remdesivir is a delayed translocation inhibitor of SARS-CoV-2 replication.

Remdesivir is a nucleoside analog approved by the US FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryo-EM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of 3 copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of 3 bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting.

The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.

Novel immunomodulatory properties of adenosine analogs promote their antiviral activity against SARS-CoV-2.

The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals, but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling, we hypothesize that antiviral adenosine analogs, like remdesivir, have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir, we here show that these properties are due to its metabolite, GS-441524, acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics.

Light-driven post-translational installation of reactive protein side chains.

Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature1,2. Although synthetic protein functionalization strategies allow mimicry of PTMs3,4, as well as formation of unnatural protein variants with diverse potential functions, including drug carrying5, tracking, imaging6 and partner crosslinking7, the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C-C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH2C• radicals that form the native (ß-CH2-γ-CH2) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe(II) generates RF2C• radicals that form equivalent ß-CH2-γ-CF2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF2-labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized 'alkylator proteins' with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function.

Reversible dougong structured receptor-ligand recognition for building dynamic extracellular matrix mimics.

Dynamic biomaterials excel at recapitulating the reversible interlocking and remoldable structure of the extracellular matrix (ECM), particularly in manipulating cell behaviors and adapting to tissue morphogenesis. While strategies based on dynamic chemistries have been extensively studied for ECM-mimicking dynamic biomaterials, biocompatible molecular means with biogenicity are still rare. Here, we report a nature-derived strategy for fabrication of dynamic biointerface as well as a three-dimensional (3D) hydrogel structure based on reversible receptor-ligand interaction between the glycopeptide antibiotic vancomycin and dipeptide d-Ala-d-Ala. We demonstrate the reversible regulation of multiple cell types with the dynamic biointerface and successfully implement the dynamic hydrogel as a functional antibacterial 3D scaffold to treat tissue repair. In view of the biogenicity and high applicability, this nature-derived reversible molecular strategy will bring opportunities for malleable biomaterial design with great potential in biomedicine.

Persulfide Transfer to SufE Activates the Half-Sites Reactivity of the E. coli Cysteine Desulfurase SufS.

The Escherichia coli cysteine desulfurase SufS (EcSufS) is a dimeric, PLP-dependent enzyme responsible for sulfur mobilization in the SUF Fe-S cluster bioassembly pathway. The enzyme uses cysteine as a sulfur source and generates alanine and a covalent persulfide located on an active site of cysteine. Optimal in vitro activity of EcSufS requires the presence of the transpersulfurase protein, EcSufE, and a strong reductant. Here, presteady-state and single-turnover kinetics are used to investigate the mechanism of EcSufS activation by EcSufE. In the absence of EcSufE, EcSufS exhibits a presteady-state burst of product production with an amplitude of ∼0.4 active site equivalents, consistent with a half-sites reactivity. KinTek Explorer was used to isolate the first turnover of alanine formation and fit the data with a simplified kinetic mechanism with steps for alanine formation (k3) and a net rate constant for the downstream steps (k5). Using this treatment, microscopic rate constants of 2.3 ± 0.5 s-1 and 0.10 ± 0.01 s-1 were determined for k3 and k5, respectively. The inclusion of EcSufE in the reaction results in a similar rate constant for k3 but induces a 10-fold enhancement of k5 to 1.1 ± 0.2 s-1, such that both steps are partially rate-determining. The most likely downstream step where EcSufE could exert influence on EcSufS activity is the removal of the persulfide intermediate. Importantly, this step appears to serve as a limiting feature in the half-sites activity such that activating persulfide transfer allows for rapid shifting between active sites. Single-turnover assays show that the presence of EcSufE slightly slowed the rates of alanine-forming steps, suggesting it does not activate steps in the desulfurase half reaction.

Tailoring Synthetic Polypeptide Design for Directed Fibril Superstructure Formation and Enhanced Hydrogel Properties.

The biological significance of self-assembled protein filament networks and their unique mechanical properties have sparked interest in the development of synthetic filament networks that mimic these attributes. Building on the recent advancement of autoaccelerated ring-opening polymerization of amino acid N-carboxyanhydrides (NCAs), this study strategically explores a series of random copolymers comprising multiple amino acids, aiming to elucidate the core principles governing gelation pathways of these purpose-designed copolypeptides. Utilizing glutamate (Glu) as the primary component of copolypeptides, two targeted pathways were pursued: first, achieving a fast fibrillation rate with lower interaction potential using serine (Ser) as a comonomer, facilitating the creation of homogeneous fibril networks; and second, creating more rigid networks of fibril clusters by incorporating alanine (Ala) and valine (Val) as comonomers. The selection of amino acids played a pivotal role in steering both the morphology of fibril superstructures and their assembly kinetics, subsequently determining their potential to form sample-spanning networks. Importantly, the viscoelastic properties of the resulting supramolecular hydrogels can be tailored according to the specific copolypeptide composition through modulations in filament densities and lengths. The findings enhance our understanding of directed self-assembly in high molecular weight synthetic copolypeptides, offering valuable insights for the development of synthetic fibrous networks and biomimetic supramolecular materials with custom-designed properties.

Competing Reaction Mechanisms of Peptide Bond Formation in Water Revealed by Deep Potential Molecular Dynamics and Path Sampling.

The formation of an amide bond is an essential step in the synthesis of materials and drugs, and in the assembly of amino acids to form peptides. The mechanism of this reaction has been studied extensively, in particular to understand how it can be catalyzed, but a representation capable of explaining all the experimental data is still lacking. Numerical simulation should provide the necessary molecular description, but the solvent involvement poses a number of challenges. Here, we combine the efficiency and accuracy of neural network potential-based reactive molecular dynamics with the extensive and unbiased exploration of reaction pathways provided by transition path sampling. Using microsecond-scale simulations at the density functional theory level, we show that this method reveals the presence of two competing distinct mechanisms for peptide bond formation between alanine esters in aqueous solution. We describe how both reaction pathways, via a general base catalysis mechanism and via direct cleavage of the tetrahedral intermediate respectively, change with pH. This result contrasts with the conventional mechanism involving a single pathway in which only the barrier heights are affected by pH. We show that this new proposal involving two competing mechanisms is consistent with the experimental data, and we discuss the implications for peptide bond formation under prebiotic conditions and in the ribosome. Our work shows that integrating deep potential molecular dynamics with path sampling provides a powerful approach for exploring complex chemical mechanisms.

Kinetic Resolution of Epimeric Proteins Enables Stereoselective Chemical Mutagenesis.

Chemical mutagenesis via dehydroalanine (Dha) is a powerful method to tailor protein structure and function, allowing the site-specific installation of post-translational modifications and non-natural functional groups. Despite the impressive versatility of this method, applications have been limited, as products are formed as epimeric mixtures, whereby the modified amino acid is present as both the desired l-configuration and a roughly equal amount of the undesired d-isomer. Here, we describe a simple remedy for this issue: removal of the d-isomer via proteolysis using a d-stereoselective peptidase, alkaline d-peptidase (AD-P). We demonstrate that AD-P can selectively cleave the d-isomer of epimeric residues within histone H3, GFP, Ddx4, and SGTA, allowing the installation of non-natural amino acids with stereochemical control. Given the breadth of modifications that can be introduced via Dha and the simplicity of our method, we believe that stereoselective chemoenzymatic mutagenesis will find broad utility in protein engineering and chemical biology applications.

Facile Method for Determining Lanthipeptide Stereochemistry.

Lanthipeptides make up a large group of natural products that belong to the ribosomally synthesized and post-translationally modified peptides (RiPPs). Lanthipeptides contain lanthionine and methyllanthionine bis-amino acids that have varying stereochemistry. The stereochemistry of new lanthipeptides is often not determined because current methods require equipment that is not standard in most laboratories. In this study, we developed a facile, efficient, and user-friendly method for detecting lanthipeptide stereochemistry, utilizing advanced Marfey's analysis with detection by liquid chromatography coupled with mass spectrometry (LC-MS). Under optimized conditions, 0.05 mg of peptide is sufficient to characterize the stereochemistry of five (methyl)lanthionines of different stereochemistry using a simple liquid chromatography setup, which is a much lower detection limit than current methods. In addition, we describe methods to readily access standards of the three different methyllanthionine stereoisomers and two different lanthionine stereoisomers that have been reported in known lanthipeptides. The developed workflow uses a commonly used nonchiral column system and offers a scalable platform to assist antimicrobial discovery. We illustrate its utility with an example of a lanthipeptide discovered by genome mining.

Enantiomer-Specific Colorimetric Tandem Assays for Salivary d-Alanine Associated with Gastric Cancer.

Salivary d-alanine (d-Ala) and d-proline (d-Pro) are of concern for their potential in the noninvasive diagnosis of gastric cancer (GC). Most reports have succeeded in determining the total concentration of d-Ala and d-Pro. However, for personalized diagnosis and better elucidation of the underlying specific correlation of d-Ala (or d-Pro) with GC, it is desirable to determine the specific concentration of d-Ala or d-Pro. Herein, we propose an enantiomer-specific tandem assay of d-Ala based on the colorimetric reaction between 2,4-dinitrophenylhydrazine and pyruvic acid generated from the deamination of d-Ala catalyzed by d-amino acid oxidase, which is easily distinguished from l-form amino acids, d-Pro, and many other species. A linear concentration range is established from 20 to 400 µmol/L with a limit of detection of 1.01 µmol/L. Real saliva sample tests reveal that the levels of d-Ala in GC cases are remarkably higher than those in healthy individuals, which offers a simple and low-cost strategy for GC diagnosis. Simultaneously, the total concentrations of d-Ala and d-Pro in saliva are determined. Hence, the concentration of d-Pro and the proportion of d-Ala could be calculated, which further provides more molecule- and individual-specific information. This research may offer a convenient method for noninvasive diagnosis of GC and pave a new route to explore the potentials of rare d-form amino acids in disease diagnosis and treatment.

The Influence of Disulfide, Thioacetal and Lanthionine-Bridges on the Conformation of a Macrocyclic Peptide.

Cyclisation of peptides by forming thioether (lanthionine), disulfide (cystine) or methylene thioacetal bridges between side chains is established as an important tool to stabilise a given structure, enhance metabolic stability and optimise both potency and selectivity. However, a systematic comparative study of the effects of differing bridging modalities on peptide conformation has not previously been carried out. In this paper, we have used the NMR deconvolution algorithm, NAMFIS, to determine the conformational ensembles, in aqueous solution, of three cyclic analogues of angiotensin(1-7), incorporating either disulfide, or non-reducible thioether or methylene thioacetal bridges. We demonstrate that the major solution conformations are conserved between the different bridged peptides, but the distribution of conformations differs appreciably. This suggests that subtle differences in ring size and bridging structure can be exploited to fine-tune the conformational properties of cyclic peptides, which may modulate their bioactivities.

Real-Time Metabolic Magnetic Resonance Spectroscopy of Pancreatic and Colon Cancer Tumor-Xenografts with Parahydrogen Hyperpolarized 1-13C Pyruvate-d3.

Parahydrogen-induced polarization (PHIP) is an emerging technique to enhance the signal of stable isotope metabolic contrast agents for Magnetic Resonance (MR). The objective of this study is to continue establishing 1-13C-pyruvate-d3, signal-enhanced via PHIP, as a hyperpolarized contrast agent, obtained in seconds, to monitor metabolism in human cancer. Our focus was on human pancreatic and colon tumor xenografts. 1-13C-vinylpyruvate-d6 was hydrogenated using parahydrogen. Thereafter, the polarization of the protons was transferred to 13C. Following a workup procedure, the free hyperpolarized 1-13C-pyruvate-d3 was obtained in clean aqueous solution. After injection into animals bearing either pancreatic or colon cancer xenografts, slice-selective MR spectra were acquired and analyzed to determine rate constants of metabolic conversion into lactate and alanine. 1-13C-pyruvate-d3 proved to follow the increased metabolic rate to lactate and alanine in the tumor xenografts.

Stabilization of Prebiotic Vesicles by Peptides Depends on Sequence and Chirality: A Mechanism for Selection of Protocell-Associated Peptides.

Cells require oligonucleotides and polypeptides with specific, homochiral sequences to perform essential functions, but it is unclear how such oligomers were selected from random sequences at the origin of life. Cells were probably preceded by simple compartments such as fatty acid vesicles, and oligomers that increased the stability, growth, or division of vesicles could have thereby increased in frequency. We therefore tested whether prebiotic peptides alter the stability or growth of vesicles composed of a prebiotic fatty acid. We find that three of 15 dipeptides tested reduce salt-induced flocculation of vesicles. All three contain leucine, and increasing their length increases the efficacy. Also, leucine-leucine but not alanine-alanine increases the size of vesicles grown by multiple additions of micelles. In a molecular simulation, leucine-leucine docks to the membrane, with the side chains inserted into the hydrophobic core of the bilayer, while alanine-alanine fails to dock. Finally, the heterochiral forms of leucine-leucine, at a high concentration, rapidly shrink the vesicles and make them leakier and less stable to high pH than the homochiral forms do. Thus, prebiotic peptide-membrane interactions influence the flocculation, growth, size, leakiness, and pH stability of prebiotic vesicles, with differential effects due to sequence, length, and chirality. These differences could lead to a population of vesicles enriched for peptides with beneficial sequence and chirality, beginning selection for the functional oligomers that underpin life.

Switchable Coacervate Formation via Amino Acid Functionalization of Poly(dehydroalanine).

Our group recently developed a family of side-chain amino acid-functionalized poly(S-alkyl-l-homocysteines), Xaa-CH (Xaa = generic amino acid), which possess the ability to form environmentally responsive coacervates in water. In an effort to further study how the molecular structure affects polypeptide coacervate formation, we prepared side-chain amino acid-functionalized poly(S-alkyl-rac-cysteines), Xaa-rac-C, via post-polymerization modification of poly(dehydroalanine), ADH. The use of the ADH platform allowed straightforward synthesis of a diverse range of side-chain amino acid-functionalized polypeptides via direct reaction of unprotected l-amino acid 2-mercaptoethylamides with ADH. Despite their differences in the main-chain structure, we found that Xaa-rac-C can form coacervates with properties similar to those seen with Xaa-CH. These results suggest that the incorporation of side-chain amino acids onto polypeptides may be a way to generally favor coacervation. The incorporation of l-methionine in Met-rac-C allowed the preparation of coacervates with improved stability against high ionic strength media. Further, the presence of additional thioether groups in Met-rac-C resulted in an increased solubility change upon oxidation allowing facile reversible redox switching of coacervate formation in aqueous media.

Indolizinylalanine Regioisomers: Tryptophan Isosteres with Bathochromic Fluorescence Emission.

We have developed a high yielding synthesis of indolizine and directly elaborated the molecule into three optically active indolizinylalanine regioisomers. The protocols exploit metal catalyzed coupling of indolizinyl-halides with organozinc reagents derived from carbamoylated iodoalanine esters. The scalable protocols provide products in a form amenable to solid-phase peptide synthesis (SPPS). When incorporated into peptides, the indolizine heterocycle is more basic and markedly less nucleophilic than tryptophan. Its protonated vinylpyridinium form is deeply colored in solution while the neutral heterocycle is highly fluorescent. The fluorescence quantum yield of indolizine exceeds that of indole and aza-indoles in water, suggesting that indolizinylalanines could be powerful optical probes of protein structure and dynamics, functioning as true tryptophan isosteres.

Systematic alanine and stapling mutational analysis of antimicrobial peptide Chem-KVL.

Chem-KVL is a tandem repeating peptide, with 14 amino acids that was modified based on a short peptide from a fragment of the human host defense protein chemerin. Chem-KVL increases cationicity and hydrophobicity and shows broad-spectrum antibacterial activity. To determine the molecular determinants of Chem-KVL and whether staple-modified Chem-KVL would improve antibacterial activity and protease stability or decrease cytotoxicity, we combined alanine and stapling scanning, and designed a series of alanine and staple-derived Chem-KVL peptides, termed Chem-A1 to Chem-A14 and SCL-1 to SCL-7. We next examined their antibacterial activity against several gram-positive and gram-negative bacteria, their proteolytic stability, and their cytotoxicity. Ala scanning of Chem-KVL suggested that both the positively charged residues (Lys and Arg) and the hydrophobic residues (Lue and Val) were critical for the antibacterial activities of Chem-KVL peptide. Of note, Chem-A4 was able to remarkably inhibit the growth of gram-positive and gram-negative bacteria when compared to the original peptide. And the antibacterial activities of stapled SCL-4 and SCL-7 were several times higher than those of the linear peptide against gram-positive and gram-negative bacteria. Stapling modification of peptides resulted in increased helicity and protein stability when compared with the linear peptide. These stapled peptides, especially SCL-4 and SCL-7, may serve as the leading compounds for further optimization and antimicrobial therapy.