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 (Mpro) 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 SARS-CoV-2. Upon viral entry, the +ssRNA genome is translated into two long polyproteins (pp1a or the frameshift-dependent pp1ab) containing all the non-structural proteins (nsps) required by the virus for immune modulation, replication, and ultimately, virion assembly. Included among these nsps is the cysteine protease Mpro (nsp5) which self-excises from the polyprotein, dimerizes, then consecutively cleaves 11 of the 15 cut site junctions found between each nsp within the polyprotein. Many structures of Mpro (often bound to various inhibitor or peptide inhibitors) have been detailed recently, including structures of Mpro bound to each of the polyprotein cleavage sequences, showing that Mpro can accommodate a wide range of targets within its active site. However, to date, kinetic characterization of the interaction of Mpro with each of its native cut site sequences remains incomplete. Here we present a robust and cost-effective FRET based system that benefits from a more consistent presentation of the substrate that is also closer in organization to the native polyprotein environment compared to previously reported FRET systems that employ chemically modified peptides. Using this system, we were able to show that while each site maintains a similar Michaelis constant, the catalytic efficiency of Mpro varies greatly between cut site sequences, suggesting a clear preference for the order of nsp processing.

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 (C2-C5a). Our lead direct-acting antiviral (DAA), C5a, is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30-50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H+-ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs (C5a) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.

Structural basis of broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.

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 (Mpro) 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 Mpro chimeras, we have determined X-ray crystallographic structures of Mpro 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). Capture of both acyl-enzyme intermediate and product-like complex forms of a P2(Leu) substrate in the native active site provides direct comparative characterization of these mechanistic steps as well as further informs the basis for enhanced product release of Mpro's own unique C-terminal P2(Phe) cleavage site to prevent autoinhibition. We characterize the underlying noncovalent interactions governing binding and specificity for this diverse set of substrates, showing remarkable plasticity for subsites beyond the anchoring P1(Gln)-P2(Leu/Val/Phe), representing together a near complete analysis of a multiprocessing viral protease. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for antiviral therapeutic development.

Crystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological C-terminal autoprocessing site.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes the disease COVID-19, produces replicase polyproteins 1a and 1ab that contain, respectively, 11 or 16 nonstructural proteins (nsp). Nsp5 is the main protease (Mpro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for subsequent viral assembly and maturation. We have determined X-ray crystallographic structures of this cysteine protease in its wild-type free active site state at 1.8 Å resolution, in its acyl-enzyme intermediate state with the native C-terminal autocleavage sequence at 1.95 Å resolution and in its product bound state at 2.0 Å resolution by employing an active site mutation (C145A). We characterize the stereochemical features of the acyl-enzyme intermediate including critical hydrogen bonding distances underlying catalysis in the Cys/His dyad and oxyanion hole. We also identify a highly ordered water molecule in a position compatible for a role as the deacylating nucleophile in the catalytic mechanism and characterize the binding groove conformational changes and dimerization interface that occur upon formation of the acyl-enzyme. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for future antiviral therapeutic development including revised molecular docking strategies based on Mpro inhibition.

Bacterial Signal Peptidases.

Signal peptidases are the membrane bound enzymes that cleave off the amino-terminal signal peptide from secretory preproteins . There are two types of bacterial signal peptidases . Type I signal peptidase utilizes a serine/lysine catalytic dyad mechanism and is the major signal peptidase in most bacteria. Type II signal peptidase is an aspartic protease specific for prolipoproteins. This chapter will review what is known about the structure, function and mechanism of these unique enzymes.

Expression and Purification of the Individual Bam Components BamB-E.

BamB, BamC, BamD, and BamE are lipoproteins that, along with the integral membrane protein BamA, form the ß-barrel assembly machinery (BAM) complex in the outer-membrane of Gram-negative bacteria. Elucidating the roles that these lipoproteins play in the ß-barrel assembly process requires both structural and functional studies that rely on milligram quantities of pure protein. Here, we describe a simple protocol for expressing individual BamB-BamE proteins in Escherichia coli and purifying them by nickel affinity and size-exclusion chromatography. This protocol yields pure proteins in amounts that are sufficient for crystallization trials, in vitro protein-protein interaction studies, NMR, and other biochemical experiments.

Structural characterization of the C3 domain of cardiac myosin binding protein C and its hypertrophic cardiomyopathy-related R502W mutant.

Human cardiac myosin binding protein C (cMyBP-C), a thick filament protein found within the sarcomere of cardiac muscle, regulates muscle contraction and is essential for proper muscle function. Hypertrophic cardiomyopathy (HCM), a genetic disease affecting 1 in 500 people, is the major cause of death in young athletes. It is caused by genetic mutations within sarcomeric proteins. Forty-two percent of the HCM-related mutations are found in cMyBP-C. Here we present the nuclear magnetic resonance-derived structural ensembles of the wild-type cMyBP-C C3 domain and its HCM-related R502W mutant. The C3 domain adopts an immunoglobulin-like fold, and mutation of the exposed Arg502 to a tryptophan does not perturb its structure, dynamics, or stability. However, the R502W mutation does alter the predicted electrostatic properties of the C3 domain. We hypothesize that this mutation, and other HCM-linked mutations found within the same domain, may directly disrupt the interaction of cMyBP-C with other sarcomeric proteins.

Structure and mechanism of Escherichia coli type I signal peptidase.

Type I signal peptidase is the enzyme responsible for cleaving off the amino-terminal signal peptide from proteins that are secreted across the bacterial cytoplasmic membrane. It is an essential membrane bound enzyme whose serine/lysine catalytic dyad resides on the exo-cytoplasmic surface of the bacterial membrane. This review discusses the progress that has been made in the structural and mechanistic characterization of Escherichia coli type I signal peptidase (SPase I) as well as efforts to develop a novel class of antibiotics based on SPase I inhibition. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Structure of signal peptide peptidase A with C-termini bound in the active sites: insights into specificity, self-processing, and regulation.

Bacterial signal peptide peptidase A (SppA) is a membrane-bound enzyme that utilizes a serine/lysine catalytic dyad mechanism to cleave remnant signal peptides within the cellular membrane. Bacillus subtilis SppA (SppABS) oligomerizes into a homo-octameric dome-shaped complex with eight active sites, located at the interface between each protomer. In this study, we show that SppABS self-processes its own C-termini. We have determined the crystal structure of a proteolytically stable fragment of SppABSK199A that has its C-terminal peptide bound in each of the eight active sites, creating a perfect circle of peptides. Substrate specificity pockets S1, S3, and S2' are identified and accommodate C-terminal residues Tyr331, Met329, and Tyr333, respectively. Tyr331 at the P1 position is conserved among most Bacillus species. The structure reveals that the C-terminus binds within the substrate-binding grooves in an antiparallel ß-sheet fashion. We show, by C-terminal truncations, that the C-terminus is not essential for oligomeric assembly. Kinetic analysis shows that a synthetic peptide corresponding to the C-terminus of SppABS competes with a fluorometric peptide substrate for the SppABS active site. A model is proposed for how the C-termini of SppA may function in the regulation of this membrane-bound self-compartmentalized protease.

The structure of cardiac troponin C regulatory domain with bound Cd2+ reveals a closed conformation and unique ion coordination.

The amino-terminal domain of cardiac troponin C (cNTnC) is an essential Ca(2+) sensor found in cardiomyocytes. It undergoes a conformational change upon Ca(2+) binding and transduces the signal to the rest of the troponin complex to initiate cardiac muscle contraction. Two classical EF-hand motifs (EF1 and EF2) are present in cNTnC. Under physiological conditions, only EF2 binds Ca(2+); EF1 is a vestigial site that has lost its function in binding Ca(2+) owing to amino-acid sequence changes during evolution. Proteins with EF-hand motifs are capable of binding divalent cations other than calcium. Here, the crystal structure of wild-type (WT) human cNTnC in complex with Cd(2+) is presented. The structure of Cd(2+)-bound cNTnC with the disease-related mutation L29Q, as well as a structure with the residue differences D2N, V28I, L29Q and G30D (NIQD), which have been shown to have functional importance in Ca(2+) sensing at lower temperatures in ectothermic species, have also been determined. The structures resemble the overall conformation of NMR structures of Ca(2+)-bound cNTnC, but differ significantly from a previous crystal structure of Cd(2+)-bound cNTnC in complex with deoxycholic acid. The subtle structural changes observed in the region near the mutations may play a role in the increased Ca(2+) affinity. The 1.4 Å resolution WT cNTnC structure, which is the highest resolution structure yet obtained for cardiac troponin C, reveals a Cd(2+) ion coordinated in the canonical pentagonal bipyramidal geometry in EF2 despite three residues in the loop being disordered. A Cd(2+) ion found in the vestigial ion-binding site of EF1 is coordinated in a noncanonical `distorted' octahedral geometry. A comparison of the ion coordination observed within EF-hand-containing proteins for which structures have been solved in the presence of Cd(2+) is presented. A refolded WT cNTnC structure is also presented.

Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water.

¹H, ¹³C and ¹5N resonance assignments and peptide binding site chemical shift perturbation mapping for the Escherichia coli redox enzyme chaperone DmsD.

Herein are reported the mainchain (1)H, (13)C and (15)N chemical shift assignments and amide (15)N relaxation data for Escherichia coli DmsD, a 23.3 kDa protein responsible for the correct folding and translocation of the dimethyl sulfoxide reductase enzyme complex. In addition, the observed amide chemical shift perturbations resulting from complex formation with the reductase subunit DmsA leader peptide support a model in which the 44 residue peptide makes extensive contacts across the surface of the DmsD protein.

The bacterial outer membrane ß-barrel assembly machinery.

ß-Barrel proteins found in the outer membrane of Gram-negative bacteria serve a variety of cellular functions. Proper folding and assembly of these proteins are essential for the viability of bacteria and can also play an important role in virulence. The ß-barrel assembly machinery (BAM) complex, which is responsible for the proper assembly of ß-barrels into the outer membrane of Gram-negative bacteria, has been the focus of many recent studies. This review summarizes the significant progress that has been made toward understanding the structure and function of the bacterial BAM complex.

Purification of a Tat leader peptide by co-expression with its chaperone.

We present a method for the purification of the 45 residue long leader peptide of Escherichia coli dimethyl sulfoxide reductase subunit A (DmsA(L)), a substrate of the twin arginine translocase, by co-expressing the leader peptide with its specific chaperone protein, DmsD. The peptide can be isolated from the soluble DmsA(L)/DmsD complex or conveniently from the lysate pellet fraction. The recombinant leader peptide is functionally intact as the peptide/chaperone complex can be reconstituted from purified DmsA(L) and DmsD. A construct with DmsA(L) fused to the N-terminus of DmsD (DmsA(L)-DmsD fusion) was created to further explore the properties of the leader peptide-chaperone interactions. Analytical size-exclusion chromatography in-line with multi-angle light scattering reveals that the DmsA(L)-DmsD fusion construct forms a dimer wherein each protomer binds the neighboring leader peptide. A model of this homodimeric interaction is presented.

Crystal structure of Bacillus subtilis signal peptide peptidase A.

Signal peptide peptidase A (SppA) is a membrane-bound self-compartmentalized serine protease that functions to cleave the remnant signal peptides left behind after protein secretion and cleavage by signal peptidases. SppA is found in plants, archaea and bacteria. Here, we report the first crystal structure of a Gram-positive bacterial SppA. The 2.4-Å-resolution structure of Bacillus subtilis SppA (SppA(BS)) catalytic domain reveals eight SppA(BS) molecules in the asymmetric unit, forming a dome-shaped octameric complex. The octameric state of SppA(BS) is supported by analytical size-exclusion chromatography and multi-angle light scattering analysis. Our sequence analysis, mutagenesis and activity assays are consistent with Ser147 serving as the nucleophile and Lys199 serving as the general base; however, they are located in different region of the protein, more than 29 Å apart. Only upon assembling the octamer do the serine and lysine come within close proximity, with neighboring protomers each providing one-half of the catalytic dyad, thus producing eight separate active sites within the complex, twice the number seen within Escherichia coli SppA (SppA(EC)). The SppA(BS) S1 substrate specificity pocket is deep, narrow and hydrophobic, but with a polar bottom. The S3 pocket, which is constructed from two neighboring proteins, is shallower, wider and more polar than the S1 pocket. A comparison of these pockets to those seen in SppA(EC) reveals a significant difference in the size and shape of the S1 pocket, which we show is reflected in the repertoire of peptides the enzymes are capable of cleaving.

Crystallographic analysis of the C-terminal domain of the Escherichia coli lipoprotein BamC.

In Gram-negative bacteria, the BAM complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one ß-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD and BamE. Here, the crystal structure of the C-terminal domain of E. coli BamC (BamC(C): Ala224-Ser343) refined to 1.5 Å resolution in space group H3 is reported. BamC(C) consists of a six-stranded antiparallel ß-sheet, three α-helices and one 3(10)-helix. Sequence and surface analysis reveals that most of the conserved residues within BamC(C) are localized to form a continuous negatively charged groove that is involved in a major crystalline lattice contact in which a helix from a neighbouring BamC(C) binds against this surface. This interaction is topologically and architecturally similar to those seen in the substrate-binding grooves of other proteins with BamC-like folds. Taken together, these results suggest that an identified surface on the C-terminal domain of BamC may serve as an important protein-binding surface for interaction with other BAM-complex components or substrates.

Synthesis and characterization of the arylomycin lipoglycopeptide antibiotics and the crystallographic analysis of their complex with signal peptidase.

Glycosylation of natural products, including antibiotics, often plays an important role in determining their physical properties and their biological activity, and thus their potential as drug candidates. The arylomycin class of antibiotics inhibits bacterial type I signal peptidase and is comprised of three related series of natural products with a lipopeptide tail attached to a core macrocycle. Previously, we reported the total synthesis of several A series derivatives, which have unmodified core macrocycles, as well as B series derivatives, which have a nitrated macrocycle. We now report the synthesis and biological evaluation of lipoglycopeptide arylomycin variants whose macrocycles are glycosylated with a deoxy-α-mannose substituent, and also in some cases hydroxylated. The synthesis of the derivatives bearing each possible deoxy-α-mannose enantiomer allowed us to assign the absolute stereochemistry of the sugar in the natural product and also to show that while glycosylation does not alter antibacterial activity, it does appear to improve solubility. Crystallographic structural studies of a lipoglycopeptide arylomycin bound to its signal peptidase target reveal the molecular interactions that underlie inhibition and also that the mannose is directed away from the binding site into solvent which suggests that other modifications may be made at the same position to further increase solubility and thus reduce protein binding and possibly optimize the pharmacokinetics of the scaffold.

Crystal structure of cardiac troponin C regulatory domain in complex with cadmium and deoxycholic acid reveals novel conformation.

The amino-terminal regulatory domain of cardiac troponin C (cNTnC) plays an important role as the calcium sensor for the troponin complex. Calcium binding to cNTnC results in conformational changes that trigger a cascade of events that lead to cardiac muscle contraction. The cardiac N-terminal domain of TnC consists of two EF-hand calcium binding motifs, one of which is dysfunctional in binding calcium. Nevertheless, the defunct EF-hand still maintains a role in cNTnC function. For its structural analysis by X-ray crystallography, human cNTnC with the wild-type primary sequence was crystallized under a novel crystallization condition. The crystal structure was solved by the single-wavelength anomalous dispersion method and refined to 2.2 Å resolution. The structure displays several novel features. Firstly, both EF-hand motifs coordinate cadmium ions derived from the crystallization milieu. Secondly, the ion coordination in the defunct EF-hand motif accompanies unusual changes in the protein conformation. Thirdly, deoxycholic acid, also derived from the crystallization milieu, is bound in the central hydrophobic cavity. This is reminiscent of the interactions observed for cardiac calcium sensitizer drugs that bind to the same core region and maintain the "open" conformational state of calcium-bound cNTnC. The cadmium ion coordination in the defunct EF-hand indicates that this vestigial calcium binding site retains the structural and functional elements that allow it to coordinate a cadmium ion. However, it is a result of, or concomitant with, large and unusual structural changes in cNTnC.

Crystal structure of ß-barrel assembly machinery BamCD protein complex.

The ß-barrel assembly machinery (BAM) complex of Escherichia coli is a multiprotein machine that catalyzes the essential process of assembling outer membrane proteins. The BAM complex consists of five proteins: one membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Here, we report the first crystal structure of a Bam lipoprotein complex: the essential lipoprotein BamD in complex with the N-terminal half of BamC (BamC(UN) (Asp(28)-Ala(217)), a 73-residue-long unstructured region followed by the N-terminal domain). The BamCD complex is stabilized predominantly by various hydrogen bonds and salt bridges formed between BamD and the N-terminal unstructured region of BamC. Sequence and molecular surface analyses revealed that many of the conserved residues in both proteins are found at the BamC-BamD interface. A series of truncation mutagenesis and analytical gel filtration chromatography experiments confirmed that the unstructured region of BamC is essential for stabilizing the BamCD complex structure. The unstructured N terminus of BamC interacts with the proposed substrate-binding pocket of BamD, suggesting that this region of BamC may play a regulatory role in outer membrane protein biogenesis.