Viruses traverse the human proteome through peptide interfaces that can be biomimetically leveraged for drug discovery.

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.

Francisella tularensis IglG Belongs to a Novel Family of PAAR-Like T6SS Proteins and Harbors a Unique N-terminal Extension Required for Virulence.

The virulence of Francisella tularensis, the etiological agent of tularemia, relies on an atypical type VI secretion system (T6SS) encoded by a genomic island termed the Francisella Pathogenicity Island (FPI). While the importance of the FPI in F. tularensis virulence is clearly established, the precise role of most of the FPI-encoded proteins remains to be deciphered. In this study, using highly virulent F. tularensis strains and the closely related species F. novicida, IglG was characterized as a protein featuring a unique α-helical N-terminal extension and a domain of unknown function (DUF4280), present in more than 250 bacterial species. Three dimensional modeling of IglG and of the DUF4280 consensus protein sequence indicates that these proteins adopt a PAAR-like fold, suggesting they could cap the T6SS in a similar way as the recently described PAAR proteins. The newly identified PAAR-like motif is characterized by four conserved cysteine residues, also present in IglG, which may bind a metal atom. We demonstrate that IglG binds metal ions and that each individual cysteine is required for T6SS-dependent secretion of IglG and of the Hcp homologue, IglC and for the F. novicida intracellular life cycle. In contrast, the Francisella-specific N-terminal α-helical extension is not required for IglG secretion, but is critical for F. novicida virulence and for the interaction of IglG with another FPI-encoded protein, IglF. Altogether, our data suggest that IglG is a PAAR-like protein acting as a bi-modal protein that may connect the tip of the Francisella T6SS with a putative T6SS effector, IglF.

Shigella deploy multiple countermeasures against host innate immune responses.

Although the intestinal epithelium is equipped with multiple defense systems that sense bacterial components, transmit alarms to the immune system, clear the bacteria, and renew the injured epithelial lining, mucosal bacterial pathogens are capable of efficiently colonizing the intestinal epithelium, because they have evolved systems that modulate the inflammatory and immune responses of the host and exploit the harmful environments as replicative niches. In this review we highlight current topics concerning Shigella's tactics that interfere with the innate immune systems.

Structural determinants underlying photoprotection in the photoactive orange carotenoid protein of cyanobacteria.

The photoprotective processes of photosynthetic organisms involve the dissipation of excess absorbed light energy as heat. Photoprotection in cyanobacteria is mechanistically distinct from that in plants; it involves the orange carotenoid protein (OCP), a water-soluble protein containing a single carotenoid. The OCP is a new member of the family of blue light-photoactive proteins; blue-green light triggers the OCP-mediated photoprotective response. Here we report structural and functional characterization of the wild type and two mutant forms of the OCP, from the model organism Synechocystis PCC6803. The structural analysis provides high resolution detail of the carotenoid-protein interactions that underlie the optical properties of the OCP, unique among carotenoid-proteins in binding a single pigment per polypeptide chain. Collectively, these data implicate several key amino acids in the function of the OCP and reveal that the photoconversion and photoprotective responses of the OCP to blue-green light can be decoupled.

Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC Component.

The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.

Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component.

The cytoplasmic membrane protein TatB is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. Together with the TatC component it forms a complex that functions as a membrane receptor for substrate proteins. Structural predictions suggest that TatB is anchored to the membrane via an N-terminal transmembrane alpha-helix that precedes an amphipathic alpha-helical section of the protein. From truncation analysis it is known that both these regions of the protein are essential for function. Here we construct 31 unique cysteine substitutions in the first 42 residues of TatB. Each of the substitutions results in a TatB protein that is competent to support Tat-dependent protein translocation. Oxidant-induced disulfide cross-linking shows that both the N-terminal and amphipathic helices form contacts with at least one other TatB protomer. For the transmembrane helix these contacts are localized to one face of the helix. Molecular modeling and molecular dynamics simulations provide insight into the possible structural basis of the transmembrane helix interactions. Using variants with double cysteine substitutions in the transmembrane helix, we were able to detect cross-links between up to five TatB molecules. Protein purification showed that species containing at least four cross-linked TatB molecules are found in correctly assembled TatBC complexes. Our results suggest that the transmembrane helices of TatB protomers are in the center rather than the periphery of the TatBC complex.