Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections.

Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.

Investigating the functional role of a buried interchain aromatic cluster in Escherichia coli GrpE dimer.

Aromatic clusters in the core of proteins are often involved in imparting structural stability to proteins. However, their functional importance is not always clear. In this study, we investigate the thermosensing role of a phenylalanine cluster present in the GrpE homodimer. GrpE, which acts as a nucleotide exchange factor for the molecular chaperone DnaK, is well known for its thermosensing activity resulting from temperature-dependent structural changes that allow control of chaperone function. Using mutational analysis, we show that an interchain phenylalanine cluster in a four-helix bundle of the GrpE homodimer assists in the thermosensing ability of the co-chaperone. Substitution of aromatic residues with hydrophobic ones in the core of the four-helix bundle reduces the thermal stability of the bundle and that of a connected coiled-coil domain, which impacts thermosensing. Cell growth assays and SEM images of the mutants show filamentous growth of Escherichia coli cells at 42°C, which corroborates with the defect in thermosensing. Our work suggests that the interchain edge-to-face aromatic cluster is important for the propagation of the structural signal from the coiled-coil domain to the four-helical bundle of GrpE, thus facilitating GrpE-mediated thermosensing in bacteria.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of S. aureus Infections.

Staphylococcus aureus is a major human pathogen responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here we describe the development of oxadiazolonebased activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologs in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling and mouse models of infection we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes (ABPs) and validate FphE as a target for development of imaging contrast agents for the rapid detection of S. aureus infections.

Novel Antibacterial Targets in Protein Biogenesis Pathways.

Antibiotic resistance has emerged as a global threat due to the ability of bacteria to quickly evolve in response to the selection pressure induced by anti-infective drugs. Thus, there is an urgent need to develop new antibiotics against resistant bacteria. In this review, we discuss pathways involving bacterial protein biogenesis as attractive antibacterial targets since many of them are essential for bacterial survival and virulence. We discuss the structural understanding of various components associated with bacterial protein biogenesis, which in turn can be utilized for rational antibiotic design. We highlight efforts made towards developing inhibitors of these pathways with insights into future possibilities and challenges. We also briefly discuss other potential targets related to protein biogenesis.

A Stutter in the Coiled-Coil Domain of Escherichia coli Co-chaperone GrpE Connects Structure with Function.

In bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a non-ideal coiled coil. Partial unfolding of this region during heat stress results in reduced nucleotide exchange and disrupts protein folding by DnaK. In this study, we elucidate the role of non-ideality in the coiled-coil domain of Escherichia coli GrpE in controlling its co-chaperone activity. The presence of a four-residue stutter introduces nonheptad periodicity in the GrpE coiled coil, resulting in global structural changes in GrpE and regulating its interaction with DnaK. Introduction of hydrophobic residues at the stutter core increased the structural stability of the protein. Using an in vitro FRET assay, we show that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, these mutants were unable to support bacterial growth at 42°C in a grpE-deleted E. coli strain. This work provides valuable insights into the functional role of a stutter in GrpE in regulating the DnaK-chaperone cycle during heat stress. More generally, our findings illustrate how stutters in a coiled-coil domain regulate structure-function trade-off in proteins.

Small-Molecule Inhibitor Prevents Insulin Fibrillogenesis and Preserves Activity.

Amyloidosis is a well-known but poorly understood phenomenon caused by the aggregation of proteins, often leading to pathological conditions. For example, the aggregation of insulin poses significant challenges during the preparation of pharmaceutical insulin formulations commonly used to treat diabetic patients. Therefore, it is essential to develop inhibitors of insulin aggregation for potential biomedical applications and for important mechanistic insights into amyloidogenic pathways. Here, we have identified a small molecule M1, which causes a dose-dependent reduction in insulin fibril formation. Biophysical analyses and docking results suggest that M1 likely binds to partially unfolded insulin intermediates. Further, M1-treated insulin had lower cytotoxicity and remained functionally active in regulating cell proliferation in cultured Drosophila wing epithelium. Thus, M1 is of great interest as a novel agent for inhibiting insulin aggregation during biopharmaceutical manufacturing.