Several families of viruses encode helicase enzymes with essential roles in viral genome replication and transcription, including several RNA virus families of pandemic concern, such as coronaviruses and flaviviruses. Viral helicases are widely considered to be promising antiviral targets with some DNA virus helicase inhibitors already approved for clinical use. Here, we demonstrate that the SARS-CoV-2 viral helicase, nsp13, is essential for viral replication. Using a previously developed in vitro helicase dsRNA unwinding assay, we screened 208 repurposed small molecules for nsp13 inhibition. Nine of these small molecules had a half-maximal inhibitory concentration (IC50) <10 µM. These molecules were tested for in vitro inhibition of nsp13 ATPase activity as well as for inhibition of Yellow Fever Virus (YFV) NS3 helicase (NS3h) activity. FPA-124, a selective inhibitor of AKT kinase, was found to inhibit the unwinding activity of both SARS-CoV-2 nsp13 and YFV NS3h, though it only inhibited ATPase activity of nsp13. Molecular ligand docking confirmed that FPA-124 likely binds within the ATP binding pocket of nsp13, but not YFV NS3h. Unfortunately, FPA-124 had a low selective index in cultured lung cells, with an IC50 against SARS-CoV-2 near the half-maximal cytotoxic concentration (CC50) in cells. Overall, we developed several high-throughput assays for antiviral drug screening against viral helicases and identified several lead scaffolds that may provide much needed tools to address future pandemics and endemic diseases with few treatment options.

Chemotaxis is an adaptive mechanism that shapes the behavior of motile bacteria in habitats characterized by fluctuating and often conflicting cues environmental (e.g. stay-or-go). Chemotactic responses are orchestrated by phosphorylation of CheY, which triggers rotational switching of the flagella. In Escherichia coli and similar taxa, CheZ is the principal CheY-P phosphatase, whereas in lineages lacking CheZ, members of the structurally distinct CheC-FliY-CheX family fulfill this role. Intriguingly, some bacteria code for CheX and CheZ, presenting a conundrum regarding their function, and the role of CheX in CheZ-containing organisms is unknown. We imposed a sustained motility constraint under conditions of looming nutrient depletion in Vibrio vulnificus, which possesses both CheX and CheZ, using the c-di-GMP effector PlzD that robustly curtails swimming motility. Our analyses revealed that the activity of CheX, but not CheZ, could be attenuated to mitigate the imposed constraint, assigning CheX a pivotal function in fine-tuning foraging behavior during a “stay-or-go” decision. V. vulnificus CheX maintained CheY-P phosphatase activity despite its conserved dimeric fold structure exhibiting divergence in active-site architecture, suggesting a preserved catalytic mechanism among distantly related homologs. Co-conservation of cheX and cheZ across disparate bacterial phyla suggests their adaptative retention confers robustness and versatility to chemotactic control. In a bacterium where they coexist, CheX tuned CheY-P–dependent motility under looming nutrient depletion, while CheZ did not, and their co-conservation likely provide robust, versatile control of stay-or-go foraging decisions across diverse bacteria.

The multidrug-resistant sepsis pathogen Acinetobacter baumannii has diverged from model γ-proteobacteria in fundamental ways, hindering efforts to develop new lines of attack against the microbe. A major area of divergence is cell division. The pathogen lacks several widely conserved division enzymes, such as FtsEX, and instead possesses a suite of atypical gene products showing no sequence similarity to any well-characterized proteins. Key among these unusual proteins is AdvA. In previous Tn-seq studies, we identified AdvA as essential for A. baumannii division and fluoroquinolone resistance. The protein comprises an N-terminal transmembrane/periplasmic region connected to a C-terminal unannotated cytoplasmic domain. Interestingly, most advA transposon insertions were lethal unless they occurred within the linker between these two regions. The roles of AdvA in cell division and the basis for positional transposon effects were unclear. Here, we combine mutagenesis with fluorescence localization, two-hybrid, and structural analyses to investigate how AdvA domains function in assembling and activating the A. baumannii divisome. We show that AdvA depletion profoundly disrupts divisome construction at Z-rings. This dependence is based on numerous interactions by AdvA with divisome proteins, with its N-terminal region binding multiple components and the cytoplasmic domain binding one (the early protein ZipA). In addition, we identified substitutions in FtsB and FtsW that suppress AdvA essentiality, consistent with a role in divisome protein activation as well as recruitment. Finally, we determined the structure of the AdvA cytoplasmic domain, which revealed a novel 3D-fold resembling adenylyl/guanylyl cyclases with striking deviations. Most notably, AdvA lacks canonical catalytic and dimerization sites and contains unusual features, including a positively charged tip promoting fluoroquinolone resistance and an extra C-terminal helix essential to divisome interactions and cell division. The critical nature of the most C-terminal structure in AdvA helps explain the positional effects of transposon insertions and facilitated identification of a distant structural homolog in the pathogen Pseudomonas aeruginosa. These results illuminate a new type of control protein governing bacterial division that could be exploited for improved antimicrobial strategies targeting nosocomial infections.

Type IV pili (T4P) mediate surface motility, host interactions, and DNA uptake through cycles of extension and retraction. While the primary retraction ATPase PilT has been extensively characterized, its homolog PilU remains less well understood despite being demonstrated as a PilT-dependent retraction ATPase. Here, we determined six PilU structures by cryo-electron microscopy and x-ray crystallography. The structures reveal a homohexameric assembly stabilized by interactions between the C-terminal catalytic domain of one subunit and the N-terminal PAS-like domain of a neighboring subunit. PilU adopts multiple conformational states, exhibiting different combinations of open and closed interfaces even in the absence of nucleotide. Comparison with PilT highlights structural features that likely underlie PilU's weak ATPase activity and its dependence on PilT for function. Together, these findings provide a structural framework for understanding PilU's role within the T4P retraction machinery.

Antibiotic resistance remains a leading cause of severe infections worldwide. Small changes in protein sequence can impact antibiotic efficacy. Here, we report deposition of 58 X-ray crystal structures of bacterial proteins that are known targets for antibiotics, which expands knowledge of structural variation to support future antibiotic discovery or modifications.

Cytochromes P450 (CYPs) form one of the largest enzyme superfamilies, with similar structural folds yet biological functions varying from synthesis of physiologically essential compounds to metabolism of myriad xenobiotics. Sterol 14α-demethylases (CYP51s) represent a very special P450 family, regarded as a possible evolutionary progenitor for all currently existing P450s. In metazoans CYP51 is critical for the biosynthesis of sterols including cholesterol. Here we determined the crystal structures of ligand-free CYP51s from the abyssal fish Coryphaenoides armatus and human-. Comparative sequence-structure-function analysis revealed specific structural elements that imply elevated conformational flexibility, uncovering a molecular basis for faster catalytic rates, lower substrate selectivity, and intrinsic resistance to inhibition. In addition, the C. armatus structure displayed a large-scale repositioning of structural segments that, in vivo, are immersed in the endoplasmic reticulum membrane and border the substrate entrance (the FG arm, >20 Å, and the β4 hairpin, >15 Å). The structural distinction of C. armatus CYP51, which is the first structurally characterized deep sea P450, suggests stronger involvement of the membrane environment in regulation of the enzyme function. We interpret this as a co-adaptation of the membrane protein structure with membrane lipid composition during evolutionary incursion to life in the deep sea.

Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vesicle that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated but rather have significant rotational flexibility. Phylogenetic analysis supports that the potential to form the disulfide bond is common across betacoronavirus isolates from many bat species and civets, but also one or both of the cysteines that form the disulfide bond are absent across isolates from bats and pangolins. The absence of these cysteines does not impact viral replication or protein translation.

Clinical and environmental isolates of Stenotrophomonas maltophilia produce an enterobactin-like siderophore that promotes bacterial growth under low-iron conditions. Although prior mutational and bioinformatic analyses indicated that most of the enzymes encoded by the S. maltophilia entCEBB'FA locus are suitably reminiscent of their counterparts in Escherichia coli and other bacteria, Stenotrophomonas EntB was unusual. In bacteria producing enterobactin-related molecules, EntB and its homologs are usually multi-domain proteins in which the amino portion acts as an isochorismatase and the carboxy domain serves as an aryl carrier protein (ArCP). However, in S. maltophilia the isochorismatase and ArCP functions are encoded by two distinct genes: entB and entB', respectively. Current mutant analysis was used to first confirm that S. maltophilia entB is needed for siderophore activity and bacterial growth in iron-depleted media. A crystal structure of S. maltophilia EntB was then obtained. The structure aligned with the N-terminal portion of EntB from E. coli and VibB from Vibrio cholerae, affirming the protein to be a single-domain isochorismatase. However, S. maltophilia EntB also aligned with the single-domain PhzD from Pseudomonas aeruginosa, which is a key enzyme involved in the biosynthesis of the antimicrobial compound phenazine. BLASTP searches indicated that entB and its neighboring genes are fully conserved amongst S. maltophilia strains but are variably present in other Stenotrophomonas species. The closest homologs to S. maltophilia EntB outside the genus were hypothetical proteins/putative isochorismatases in some Gram-negative bacteria (for example Pseudomonas spp. and Xanthomonas spp.), Gram-positive bacteria (Streptomyces spp. and Bacillus subtilis) and fungi (for example Rhizopus arrhizus and Knufia peltigerae).

ABSTRACT During infection, bacterial pathogens rely on secreted virulence factors to manipulate the host cell. However, in gram-positive bacteria, the molecular mechanisms underlying the folding and activity of these virulence factors after membrane translocation are not clear. Here, we solved the protein structures of two secreted parvulin and two secreted cyclophilin-like peptidyl-prolyl isomerase (PPIase) ATP-independent chaperones found in gram-positive streptococcal species. The extracellular parvulin-type PPIase, PrsA in Streptococcus pneumoniae and Streptococcus mutans maintain dimeric crystal structures reminiscent of folding catalysts that consist of two domains, a PPIase and foldase domain. Structural comparison of the two cyclophilin-like extracellular chaperones from S. pneumoniae and Streptococcus pyogenes with other cyclophilins demonstrates that this group of cyclophilin-like chaperones has novel structural appendages formed by 9- and 24-residue insertions. Furthermore, we demonstrate that deletion of prsA and slrA genes impairs the secretion of the cholesterol-dependent pore-forming toxin, pneumolysin in S. pneumoniae. Using protein pull-down and biophysical assays, we demonstrate a direct interaction between PrsA and SlrA with Ply. Then, we developed chaperone-assisted folding assays that show that the S. pneumoniae PrsA and SlrA extracellular chaperones accelerate pneumolysin folding. In addition, we demonstrate that SlrA and, for the first time, S. pyogenes PpiA exhibit PPIase activity and can bind the immunosuppressive drug, cyclosporine A. Altogether, these findings suggest a mechanistic role for streptococcal PPIase chaperones in the activity and folding of secreted virulence factors such as pneumolysin.IMPORTANCEStreptococcal species are a leading cause of lower respiratory infections that annually affect millions of people worldwide. During infection, streptococcal species secrete a medley of virulence factors that allow the bacteria to colonize and translocate to deeper tissues. In many gram-positive bacteria, virulence factors are secreted from the cytosol across the bacterial membrane in an unfolded state. The bacterial membrane-cell wall interface is exposed to the potentially harsh extracellular environment, making it difficult for native virulence factors to fold before being released into the host. ATP-independent PPIase-type chaperones, PrsA and SlrA, are thought to facilitate folding and stabilization of several unfolded proteins to promote the colonization and spread of streptococci. Here, we present crystal structures of the molecular chaperones of PrsA and SlrA homologs from streptococcal species. We provide evidence that the Streptococcus pyogenes SlrA homolog, PpiA, has PPIase activity and binds to cyclosporine A. In addition, we show that Streptococcus pneumoniae PrsA and SlrA directly interact and fold the cholesterol-dependent pore-forming toxin and critical virulence determinant, pneumolysin.

Vibrio cholerae motility is mediated by a single polar flagellum, composed of four flagellin subunits (FlaABCD) in the filament; however, only FlaA is required for motility. Class III flagellar genes, which include flaA, are controlled by the two-component FlrBC system. FlrB is a histidine kinase that phosphorylates FlrC, which activates Class III promoters. The signal(s) that control phosphotransfer between FlrB and FlrC are unknown. A V. cholerae strain lacking the “non-essential” flagellin genes (ΔflaCEDB) is non-motile. Selection for spontaneous motile strains resulted in mutations localized to a Per-Arnt-Sim (PAS) domain in the FlrB N-terminus, including the mutation L36F. The X-ray crystal structure of FlrB revealed an asymmetric dimer with a unique fold of the PAS domain. Transcriptome analysis showed that class III transcription is increased with the addition of the L36F PAS mutation to FlrB, while class III gene transcription was eliminated with a mutation at the site of phosphorylation (H135N). H135N prevents phosphorylation of purified FlrB, whereas L36F increases phosphorylation, indicating these mutations represent “off” and “on” forms of FlrB. FlrB localizes to the V. cholerae cell pole, and localization is dependent on the flagellar polar targeting protein FlhF. V. cholerae strains containing either “off” or “on” forms of FlrB were defective for intestinal colonization in infant mice (10- to 40-fold defect). Our results demonstrate that the PAS domain controls FlrB activity and class III flagellar gene expression, and FlrB must switch between inactive and active forms in order for V. cholerae to successfully colonize the intestine. IMPORTANCE Vibrio cholerae causes the severe diarrheal disease cholera when it colonizes the human intestine. The bacteria are able to swim due to a polar flagellum, and motility is linked to disease, as well as environmental persistence. This study demonstrates that FlrB, a key regulatory protein, localizes to the cell pole and controls flagellar gene transcription via a PAS domain that regulates autophosphorylation. The ability of FlrB to switch between active and inactive forms is critical for motility, as well as intestinal colonization, emphasizing the importance of V. cholerae swimming for its ability to cause disease.

Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vesicle that houses the replication–transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated but rather have significant rotational flexibility. Phylogenetic analysis supports that the potential to form the disulfide bond is common across betacoronavirus isolates from many bat species and civets, but also one or both of the cysteines that form the disulfide bond are absent across isolates from bats and pangolins. The absence of these cysteines does not impact viral replication or protein translation.

To address the ongoing threat of SARS-CoV-2 and potential emergence of novel coronaviruses, we employed a comprehensive strategy to identify and synthesize inhibitors of coronavirus methyltransferases with chemical analogs of S-adenosylhomocysteine (SAH). Two analogs, designated 4h and 4p, inhibit both mouse hepatitis virus and SARS-CoV-2 replication. Compound 4p was the most potent with half-maximal inhibition of biochemical activity at 0.2 μM and antiviral activity at ∼20 μM. This compound also has low cytotoxicity and preferentially inhibits nsp14 over nsp16 and human methyltransferases. Furthermore, molecular docking based on a newly determined crystal structure of the apo nsp16-nsp10 complex predicts that 4p occupies both the S-adenosylmethione and Gppp binding pockets of nsp14 and nsp16. Selectivity of 4p for nsp14 is likely due to the enhanced structural stability of the nsp14 binding pocket relative to nsp16. These findings highlight SAH analogs as scaffolds for pan-coronavirus therapeutics and underscore the value of structure-guided design in antiviral drug discovery.

Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli, indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCEStenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa. We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

There is an urgent need for new antibiotics given the rise of antibiotic resistance, and succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) has emerged as a promising bacterial enzyme target. DapE from Haemophilus influenzae (HiDapE) has been studied and inhibitors identified, but it is essential to explore DapE from different species to assess selective versus broad-spectrum therapeutics. We have determined the structure of DapE from the ESKAPE pathogen Acinetobacter baumannii (AbDapE) and studied inhibition by known inhibitors of HiDapE. AbDapE is inhibited by captopril and sulfate comparable to HiDapE, but AbDapE was not significantly inhibited by a known indoline sulfonamide HiDapE inhibitor. Captopril and sulfate both stabilize HiDapE by increasing the thermal melting temperature (Tm) in thermal shift assays. By contrast, sulfate decreases the stability of the AbDapE enzyme, whereas captopril increases the stability. Further, we report two crystal structures of selenomethionine-substituted AbDapE in the closed conformation, one with AbDapE in complex with succinate derived from enzymatic hydrolysis of N6-methyl-l,l-SDAP substrate and acetate (PDB code 7T1Q, 2.25 Å resolution), and a crystal structure of AbDapE with bound succinate along with l-(S)-lactate, a product of degradation of citric acid from the crystallization buffer during X-ray irradiation (PDB code 8F8O, 2.10 Å resolution).

Vibrio vulnificus causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host Ra s-related proteins in b rain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.

Cytochromes P450 (CYP) form one of the largest enzyme superfamilies on Earth, with similar structural fold but biological functions varying from synthesis of physiologically essential compounds to metabolism of myriad xenobiotics. Here we determined the crystal structures of Coryphaenoides armatus and human sterol 14α-demethylases (CYP51s). Both structures reveal elements that imply elevated conformational flexibility, uncovering molecular basis for faster catalytic rates, lower substrate selectivity, and resistance to inhibition. These elements as well as the unique inward/outward location of the FG arm/β4 hairpin in the fish CYP51 structure were not predicted by artificial intelligence molecular modelling. The structural distinction of C. armatus CYP51, which is the first structurally characterized deep-sea P450, suggests stronger involvement of the membrane environment in regulation of this enzyme function. We interpret this as a co-adaptation of membrane protein structure with changes in membrane lipid composition during evolutionary incursion to life in the deep sea.

Clostridioides difficile causes life-threatening gastrointestinal infections. It is a high-risk pathogen due to a lack of effective treatments, antimicrobial resistance, and a poorly conserved genomic core. Herein, we report 30 X-ray structures from a structure genomics pipeline spanning 13 years, representing 10.2% of the X-ray structures for this important pathogen.