Synergistic Multi-Pronged Interactions Mediate the Effective Inhibition of Alpha-Synuclein Aggregation by the Chaperone HtrA1.

Unlabelled: The misfolding, aggregation, and the seeded spread of alpha synuclein (α-Syn) aggregates are linked to the pathogenesis of various neurodegenerative diseases, including Parkinson's disease (PD). Understanding the mechanisms by which chaperone proteins prevent the production and seeding of α-Syn aggregates is crucial for developing effective therapeutic leads for tackling neurodegenerative diseases. We show that a catalytically inactive variant of the chaperone HtrA1 (HtrA1*) effectively inhibits both α-Syn monomer aggregation and templated fibril seeding, and demonstrate that this inhibition is mediated by synergistic interactions between its PDZ and Protease domains and α-Syn.

Prediction and design of protease enzyme specificity using a structure-aware graph convolutional network.

Site-specific proteolysis by the enzymatic cleavage of small linear sequence motifs is a key posttranslational modification involved in physiology and disease. The ability to robustly and rapidly predict protease-substrate specificity would also enable targeted proteolytic cleavage by designed proteases. Current methods for predicting protease specificity are limited to sequence pattern recognition in experimentally derived cleavage data obtained for libraries of potential substrates and generated separately for each protease variant.

Genetic Encoding of Arylazopyrazole Phenylalanine for Optical Control of Translation.

An arylazopyrazole was explored for its use as an enhanced photoswitchable amino acid in genetic code expansion. This new unnatural amino acid was successfully incorporated into proteins in both bacterial and mammalian cells. While photocontrol of translation required pulsed irradiations, complete selectivity for the -configuration by the pyrrolysyl tRNA synthetase was observed, demonstrating expression of a gene of interest selectively controlled via light exposure.

Modeling of ACE2 and antibodies bound to SARS-CoV-2 provides insights into infectivity and immune evasion.

Given the COVID-19 pandemic, there is interest in understanding ligand-receptor features and targeted antibody-binding attributes against emerging SARS-CoV-2 variants. Here, we developed a large-scale structure-based pipeline for analysis of protein-protein interactions regulating SARS-CoV-2 immune evasion. First, we generated computed structural models of the Spike protein of 3 SARS-CoV-2 variants (B.

Multifaceted interactions mediated by intrinsically disordered regions play key roles in alpha synuclein aggregation.

The aggregation of Alpha Synuclein (α-Syn) into fibrils is associated with the pathology of several neurodegenerative diseases. Pathologic aggregates of α-Syn adopt multiple fibril topologies and are known to be transferred between cells via templated seeding. Monomeric α-Syn is an intrinsically disordered protein (IDP) with amphiphilic N-terminal, hydrophobic-central, and negatively charged C-terminal domains.

Computational design of nanomolar-binding antibodies specific to multiple SARS-CoV-2 variants by engineering a specificity switch of antibody 80R using RosettaAntibodyDesign (RAbD) results in potential generalizable therapeutic antibodies for novel SARS-CoV-2 virus.

The human infectious disease COVID-19 caused by the SARS-CoV-2 virus has become a major threat to global public health. Developing a vaccine is the preferred prophylactic response to epidemics and pandemics. However, for individuals who have contracted the disease, the rapid design of antibodies that can target the SARS-CoV-2 virus fulfils a critical need.

A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly.

Precise metal-protein coordination by design remains a considerable challenge. Polydentate, high-metal-affinity protein modifications, both chemical and recombinant, can enable metal localization. However, these constructs are often bulky, conformationally and stereochemically ill-defined, or coordinately saturated.

Monomer Choice Influences -Acryloyl Amino Acid Grafter Conversion via Protease Catalysis.

End-capped peptides modified with reactive functional groups on the N-terminus provide a route to prepare peptide-polymer conjugates for a broad range of applications. Unfortunately, current chemical methods to construct modified peptides rely largely on solid-phase peptide synthesis (SPPS), which lacks green preparative characteristics and is costly, thus limiting its applicability to specialty applications such as regenerative medicine. This work evaluates N-terminally modified -acryloyl-glutamic acid diethyl ester, -acryloyl-leucine ethyl ester, and -acryloyl-alanine ethyl ester as grafters and papain as the protease for the direct addition of amino acid ethyl ester (AA-OEt) monomers via protease-catalyzed peptide synthesis (PCPS) and the corresponding formation of -acryloyl-functionalized oligopeptides in a one-pot aqueous reaction.

Prediction and Design of Protease Enzyme Specificity Using a Structure-Aware Graph Convolutional Network.

Site-specific proteolysis by the enzymatic cleavage of small linear sequence motifs is a key post-translational modification involved in physiology and disease. The ability to robustly and rapidly predict protease substrate specificity would also enable targeted proteolytic cleavage - editing - of a target protein by designed proteases. Current methods for predicting protease specificity are limited to sequence pattern recognition in experimentally-derived cleavage data obtained for libraries of potential substrates and generated separately for each protease variant.

A comprehensive survey of coronaviral main protease active site diversity in 3D: Identifying and analyzing drug discovery targets in search of broad specificity inhibitors for the next coronavirus pandemic.

Although the rapid development of therapeutic responses to combat SARS-CoV-2 represents a great human achievement, it also demonstrates untapped potential for advanced pandemic preparedness. Cross-species efficacy against multiple human coronaviruses by the main protease (MPro) inhibitor nirmatrelvir raises the question of its breadth of inhibition and our preparedness against future coronaviral threats. Herein, we describe sequence and structural analyses of 346 unique MPro enzymes from all coronaviruses represented in the NCBI Virus database.

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution, new validation tools, and implications for the future.

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) "Resolution Revolution" made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps.

Highly stereoselective and enantiodivergent synthesis of cyclopropylphosphonates with engineered carbene transferases.

Organophosphonate compounds have represented a rich source of biologically active compounds, including enzyme inhibitors, antibiotics, and antimalarial agents. Here, we report the development of a highly stereoselective strategy for olefin cyclopropanation in the presence of a phosphonyl diazo reagent as carbene precursor. In combination with a 'substrate walking' protein engineering strategy, two sets of efficient and enantiodivergent myoglobin-based biocatalysts were developed for the synthesis of both (1,2) and (1,2) enantiomeric forms of the desired cyclopropylphosphonate ester products.

Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign.

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, internucleotidyl O-P linkages. Can this linkage selectivity be overcome by design to produce xenonucleic acids? Here, we report that the structure-guided redesign of an archaeal DNA polymerase, 9°N, exhibits a new activity undetectable in the wild-type enzyme: catalyzing the formation of internucleotidyl N-P linkages using 3'-NH-ddNTPs. Replacing a metal-binding aspartate in the 9°N active site with asparagine was key to the emergence of this unnatural enzyme activity.

Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization.

Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether "staples" into a protein via cysteine alkylation with the noncanonical amino acid -2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy.

Genetic and Structural Analysis of SARS-CoV-2 Spike Protein for Universal Epitope Selection.

Evaluation of immunogenic epitopes for universal vaccine development in the face of ongoing SARS-CoV-2 evolution remains a challenge. Herein, we investigate the genetic and structural conservation of an immunogenically relevant epitope (C662-C671) of spike (S) protein across SARS-CoV-2 variants to determine its potential utility as a broad-spectrum vaccine candidate against coronavirus diseases. Comparative sequence analysis, structural assessment, and molecular dynamics simulations of C662-C671 epitope were performed.

Massively parallel, computationally guided design of a proenzyme.

Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design.

Enantioselective Synthesis of α-Trifluoromethyl Amines via Biocatalytic N-H Bond Insertion with Acceptor-Acceptor Carbene Donors.

The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N-H carbene insertion reaction catalyzed by engineered variants of cytochrome from . Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor.

Structural models of SARS-CoV-2 Omicron variant in complex with ACE2 receptor or antibodies suggest altered binding interfaces.

There is enormous ongoing interest in characterizing the binding properties of the SARS-CoV-2 Omicron Variant of Concern (VOC) (B.1.1.

Ensuring scientific reproducibility in bio-macromolecular modeling via extensive, automated benchmarks.

Each year vast international resources are wasted on irreproducible research. The scientific community has been slow to adopt standard software engineering practices, despite the increases in high-dimensional data, complexities of workflows, and computational environments. Here we show how scientific software applications can be created in a reproducible manner when simple design goals for reproducibility are met.

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first 6 months of the COVID-19 pandemic.

Understanding the molecular evolution of the SARS-CoV-2 virus as it continues to spread in communities around the globe is important for mitigation and future pandemic preparedness. Three-dimensional structures of SARS-CoV-2 proteins and those of other coronavirusess archived in the Protein Data Bank were used to analyze viral proteome evolution during the first 6 months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48 000 viral isolates revealed how each one of 29 viral proteins have undergone amino acid changes.

Computational structure prediction provides a plausible mechanism for electron transfer by the outer membrane protein Cyc2 from Acidithiobacillus ferrooxidans.

Cyc2 is the key protein in the outer membrane of Acidithiobacillus ferrooxidans that mediates electron transfer between extracellular inorganic iron and the intracellular central metabolism. This cytochrome c is specific for iron and interacts with periplasmic proteins to complete a reversible electron transport chain. A structure of Cyc2 has not yet been characterized experimentally.

MHCEpitopeEnergy, a Flexible Rosetta-Based Biotherapeutic Deimmunization Platform.

As non-"self" macromolecules, biotherapeutics can trigger an immune response that can reduce drug efficacy, require patients to be taken off therapy, or even cause life-threatening reactions. To enable the flexible and facile design of protein biotherapeutics while reducing the prevalence of T-cell epitopes that drive immune recognition, we have integrated into the Rosetta protein design suite a new scoring term that allows design protocols to account for predicted or experimentally identified epitopes in the optimized objective function. This flexible scoring term can be used in any Rosetta design trajectory, can be targeted to specific regions of a protein, and can be readily extended to work with a variety of epitope predictors.

Biomolecular Condensates: Sequence Determinants of Phase Separation, Microstructural Organization, Enzymatic Activity, and Material Properties.

This perspective article highlights recent progress and emerging challenges in understanding the formation and function of membraneless organelles (MLOs). A long-term goal in the MLO field is to identify the sequence-encoded rules that dictate the formation of compositionally controlled biomolecular condensates, which cells utilize to perform a wide variety of functions. The molecular organization of the different components within a condensate can vary significantly, ranging from a homogeneous mixture to core-shell droplet structures.

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic.

Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores.