Kinetic comparison of all eleven viral polyprotein cleavage site processing events by SARS-CoV-2 main protease using a linked protein FRET platform.

The main protease (M) remains an essential therapeutic target for COVID-19 post infection intervention given its critical role in processing the majority of viral proteins encoded by the genome of severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2). Upon viral entry, the +ssRNA genome is translated into two long polyproteins (pp1a or the frameshift-dependent pp1ab) containing all the nonstructural proteins (nsps) required by the virus for immune modulation, replication, and ultimately, virion assembly. Included among these nsps is the cysteine protease M (nsp5) which self-excises from the polyprotein, dimerizes, then sequentially cleaves 11 of the 15 cut-site junctions found between each nsp within the polyprotein.

A novel class of broad-spectrum active-site-directed 3C-like protease inhibitors with nanomolar antiviral activity against highly immune-evasive SARS-CoV-2 Omicron subvariants.

Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors (). Our lead direct-acting antiviral (DAA), , is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro.

Characterization of the structure and self-assembly of two distinct class IB hydrophobins.

Hydrophobins are small proteins secreted by fungi that accumulate at interfaces, modify surface hydrophobicity, and self-assemble into large amyloid-like structures. These unusual properties make hydrophobins an attractive target for commercial applications as emulsifiers and surface modifying agents. Hydrophobins have diverse sequences and tertiary structures, complicating attempts to characterize how they function.

X-ray crystallographic characterization of the SARS-CoV-2 main protease polyprotein cleavage sites essential for viral processing and maturation.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, produces polyproteins 1a and 1ab that contain, respectively, 11 or 16 non-structural proteins (nsp). Nsp5 is the main protease (M) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for viral assembly and maturation. Using C-terminally substituted M chimeras, we have determined X-ray crystallographic structures of M in complex with 10 of its 11 viral cleavage sites, bound at full occupancy intermolecularly in trans, within the active site of either the native enzyme and/or a catalytic mutant (C145A).

Expression, purification, and refolding of diverse class IB hydrophobins.

Hydrophobins are low molecular weight proteins secreted by fungi that are extremely surface-active and able to self-assemble into larger structures. Due to their unusual biochemical properties, hydrophobins are an attractive target for commercial applications such as drug emulsification and surface modification. When produced in E.

Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct F Labels.

To probe ligand-receptor binding at the atomic-level, a frequent approach involves multidimensional nuclear magnetic resonance (NMR) spectroscopy experiments relying on C- and/or N-enrichment alongside H. Alternatively, the lack of fluorine in biomolecules may be exploited through specific incorporation of F nuclei into a sample. The F nucleus is highly sensitive to environmental changes and allows for one-dimensional NMR spectroscopic study, with perturbation to chemical shift and spin dynamics diagnostic of structural change, ligand binding, and modified conformational sampling.

Apelinergic System Structure and Function.

Apelin and apela (ELABELA/ELA/Toddler) are two peptide ligands for a class A G-protein-coupled receptor named the apelin receptor (AR/APJ/APLNR). Ligand-AR interactions have been implicated in regulation of the adipoinsular axis, cardiovascular system, and central nervous system alongside pathological processes. Each ligand may be processed into a variety of bioactive isoforms endogenously, with apelin ranging from 13 to 55 amino acids and apela from 11 to 32, typically being cleaved C-terminal to dibasic proprotein convertase cleavage sites.

Mixed Fluorotryptophan Substitutions at the Same Residue Expand the Versatility of F Protein NMR Spectroscopy.

The strategy of applying fluorine NMR to characterize ligand binding to a membrane protein prepared with mixtures of tryptophans substituted with F at different positions on the indole ring was tested. The F NMR behavior of 4-, 5-, 6-, and 7-fluorotryptophan were directly compared as a function of both micellar environment and fragment size for two overlapping apelin receptor (AR/APJ) segments; one with a single transmembrane (TM) helix and two tryptophan residues, the other with three TM helices and two additional tryptophan residues. Chemical shifts, peak patterns, and nuclear spin relaxation rates were observed to vary as a function of micellar conditions and F substitution position in the indole ring, with the exposure of a given residue to micelle or solvent being the primary differentiating factor.

Bioactivity of the putative apelin proprotein expands the repertoire of apelin receptor ligands.

Background: Apelin is a peptide ligand for a class A G-protein coupled receptor called the apelin receptor (AR or APJ) that regulates angiogenesis, the adipoinsular axis, and cardiovascular functions. Apelin has been shown to be bioactive as 13, 17, and 36 amino acid isoforms, C-terminal fragments of the putatively inactive 55-residue proprotein (proapelin or apelin-55). Although intracellular proprotein processing has been proposed, isolation of apelin-55 from colostrum and milk demonstrates potential for secretion prior to processing and the possibility of proapelin-AR interaction.