Crystal structures and biochemical characterization of DNA sliding clamps from three Gram-negative bacterial pathogens

Abstract

Bacterial sliding clamps bind to DNA and act as protein-protein interaction hubs for several proteins involved in DNA replication and repair. The partner proteins all bind to a common pocket on sliding clamps via conserved linear peptide sequence motifs, which suggest the pocket as an attractive target for development of new antibiotics. Herein we report the X-ray crystal structures and biochemical characterization of β sliding clamps from the Gram-negative pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae. The structures reveal close similarity between the pathogen and Escherichia coli clamps and similar patterns of binding to linear clamp-binding motif peptides. The results suggest that linear motifsliding clamp interactions are well conserved and an antibiotic targeting the sliding clamp should have broadspectrum activity against Gram-negative pathogens. Crystal structures and biochemical characterization of DNA sliding clamps from three Gramnegative bacterial pathogens, Journal of Structural Biology (2018), doi: https://doi.ABSTRACT Bacterial sliding clamps bind to DNA and act as protein-protein interaction hubs for several proteins involved in DNA replication and repair. The partner proteins all bind to a common pocket on sliding clamps via conserved linear peptide sequence motifs, which suggest the pocket as an attractive target for development of new antibiotics. Herein we report the X-ray crystal structures and biochemical characterization of  sliding clamps from the Gram-negative pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae. The structures reveal close similarity between the pathogen and Escherichia coli clamps and similar patterns of binding to linear clamp-binding motif peptides. The results suggest that linear motif-sliding clamp interactions are well conserved and an antibiotic targeting the sliding clamp should have broadspectrum activity against Gram-negative pathogens.

The methylation-independent mismatch repair machinery in Pseudomonas aeruginosa

“…In canonical MMR systems such as those in E. coli and B. subtilis , MutS is thought to interact with the β-sliding clamp of the replisome complex, thereby facilitating scanning, and tightly coupling (both spatially and temporally) MMR with replication [ 20, 57–60 ]. MutS does this because it contains a conserved ‘clamp binding motif’ (CBM) which binds tightly to a discrete pocket on the β-clamp [ 57 ]. It has been suggested that this association of MutS PA with β PA may impede interaction of MutS PA with MutL PA [ 55 ].…”

“…||Though absent in Staphylococcus aureus RF122, a MutL homolog is present in strain MSSA476. Continued replication[20,[57][58][59][60]. MutS does this because it contains a conserved 'clamp binding motif ' (CBM) which binds tightly to a discrete pocket on the β-clamp[57].…”

“…Continued replication[20,[57][58][59][60]. MutS does this because it contains a conserved 'clamp binding motif ' (CBM) which binds tightly to a discrete pocket on the β-clamp[57]. It has been suggested that this association of MutS PA with β PA may impede interaction of MutS PA with MutL PA…”

A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth

“…15 The conserved structure of SCs across Gram-positive and Gram-negative bacteria make it a readily generalizable strategy for generating other ncAA dependent auxotrophs. 16,17 To construct an orthogonal β-subunit interface, we first disrupted the native E. coli SC (SC Eco ) interface by incorporating the large and structurally distinct ncAA, pBzF, at the dimer interface, which resulted in a loss of cell viability. We then performed a selection with a library of residues around the pBzF side chain to restore its SC function.…”

“…SC plays a critical role in the processivity of DNA replication and is essential for bacterial survival. − The SC forms a head-to-tail homodimer, with each monomer having identical chain topology . The conserved structure of SCs across Gram-positive and Gram-negative bacteria make it a readily generalizable strategy for generating other ncAA dependent auxotrophs. , …”