There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2′-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP 6 ). In this study, we demonstrated that InsP 6 is not simply an allosteric cofactor, but rather binding of InsP 6 stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-Å crystal structure of this InsP 6 -bound unprocessed form of CPD was determined and revealed the scissile bond Leu 3428 -Ala 3429 captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP 6 , but was reactivated for high affinity binding of InsP 6 by cooperative binding of both a new substrate and InsP 6 . Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.Multifunctional-autoprocessing repeats-in-toxin (MARTX) 3 toxins are a family of large bacterial protein toxins with conserved repeat regions at the N and C termini that are predicted to transfer effector domains located between the repeats across the eukaryotic cell plasma membrane (1). The best characterized MARTX is the Ͼ450-kDa secreted virulence-associated MARTX of Vibrio cholerae. This toxin causes disassembly of the actin cytoskeleton and enhances V. cholerae colonization of the small intestine, possibly by facilitating evasion of phagocytic cells (2, 3). The central region of the V. cholerae MARTX toxin contains four discrete domains: the actin cross-linking domain (ACD) that introduces lysine-glutamate cross-links between actin protomers (4, 5), the Rho-inactivating domain (RID) that disables small Rho GTPases (6), an ␣␤ hydrolase of unknown function (1), and an autoprocessing cysteine protease domain (CPD) (7,8).The CPD is a 25-kDa domain found in all MARTX toxins located just before the start of the C-terminal repeats (7,8). This domain is activated for autoproteolysis upon binding inositol hexakisphosphate (InsP 6 ) (7), a molecule ubiquitously present in eukaryotic cell cytosol (9 -11), but absent in extracellular spaces and bacteria. Thus, autocatalytic processing would not occur until after translocation of the CPD and effector domains is completed. In the context of the holotoxin, catalytic residue Cys 3568 was found to be essential for the toxin to induce efficient actin cross-linking by the ACD and Rho inactivation by the RID, demonstrating that autoprocessing is essential for MARTX to induce cell rounding (8).While it is clear that InsP 6 activates the CPD and that auto...

Background: The serine-rich repeat glycoproteins Srr1 and Srr2 are surface adhesins of Streptococcus agalactiae important for pathogenicity.Results: Both Srrs bind tandem repeats of the fibrinogen Aα chain, but Srr2 has greater affinity explained by structure-function analysis of the Srrs.Conclusion: A dock, lock, and latch mechanism describes the Srr-fibrinogen interaction.Significance: The higher affinity of Srr2 may contribute to the hypervirulence of Srr2-expressing strains.

SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,

Emerging evidence suggests thePseudomonas aeruginosaaccessory genome is enriched with uncharacterized virulence genes. Identification and characterization of such genes may reveal novel pathogenic mechanisms used by particularly virulent isolates. Here, we utilized a mouse bacteremia model to quantify the virulence of 100 individualP. aeruginosabloodstream isolates and performed whole-genome sequencing to identify accessory genomic elements correlated with increased bacterial virulence. From this work, we identified a specific contact-dependent growth inhibition (CDI) system enriched among highly virulentP. aeruginosaisolates. CDI systems contain a large exoprotein (CdiA) with a C-terminal toxin (CT) domain that can vary between different isolates within a species. Prior work has revealed that delivery of a CdiA-CT domain upon direct cell-to-cell contact can inhibit replication of a susceptible target bacterium. Aside from mediating interbacterial competition, we observed our virulence-associated CdiA-CT domain to promote toxicity against mammalian cells in culture and lethality during mouse bacteremia. Structural and functional studies revealed this CdiA-CT domain to have in vitro tRNase activity, and mutations that abrogated this tRNAse activity in vitro also attenuated virulence. Furthermore, CdiA contributed to virulence in mice even in the absence of contact-dependent signaling. Overall, our findings indicate that thisP. aeruginosaCDI system functions as both an interbacterial inhibition system and a bacterial virulence factor against a mammalian host. These findings provide an impetus for continued studies into the complex role of CDI systems inP. aeruginosapathogenesis.