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.

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.

Molecular insights into the mechanism of substrate binding and catalysis of bifunctional FAD synthetase from Staphylococcus aureus.

Flavin adenine dinucleotide synthetase (FADS), a bifunctional prokaryotic enzyme, is involved in the synthesis of two vital cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Here, we investigated the biochemical characteristics of FADS from Staphylococcus aureus (Sa), a pathogenic bacteria causing food-borne diseases. The SaFADS possesses riboflavin kinase (RFK) and FMN adenylyltransferase (FMNAT) activities that transforms riboflavin to FMN and FMN to FAD, respectively. The FMNAT domain also exhibits reversible FAD pyrophosphorylase activity (FADpp). Further, we show that the FMNAT and FADpp activities are dependent on the reducing environment. Mutations of the conserved K289 and F290 residues present on the RFK domain affect the kinetic parameters of both the RFK and FMNAT domains. Additionally, the molecular dynamics analysis of apo and riboflavin: ATP: Mg2+ ternary complex of SaFADS shows that F290 is involved in stabilizing the active site geometry to hold the enzyme-substrate complex. In addition, the deletion of the αh2 helix that acts as a connecting linker between the FMNAT and RFK domains showed substantial loss of their activities. The helix deletion could have affected the flap motion of L2c, L4c, ß4n and L3n present in the close proximity resulting in the distortion of the active site geometry. In conclusion, our study has characterized the RFK and FMNAT activities of SaFADS and shown the importance of conserved K289 and F290 in RFK activity. As FADSs are potential drug targets, understanding their mechanism of action might help in discovering species-specific antibacterial drugs.