Abstract
Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drug development. It is therefore essential to decipher the polymerase-clamp binding mode across various bacterial species. Here, two residues of the E. coli clamp binding pocket, EcS346 and EcM362, and their cognate residues in M. tuberculosis and B. subtilis clamps, were mutated. The effects of these mutations on the interaction of a model peptide with these variant clamps were evaluated by thermodynamic, molecular dynamics, X-rays crystallography, and biochemical analyses. EcM362 and corresponding residues in Gram positive clamps occupy a strategic position where a mobile residue is essential for an efficient peptide interaction. EcS346 has a more subtle function that modulates the pocket folding dynamics, while the equivalent residue in B. subtilis is essential for polymerase activity and might therefore be a Gram positive-specific molecular marker. Finally, the peptide binds through an induced-fit process to Gram negative and positive pockets, but the complex stability varies according to a pocket-specific network of interactions.
Highlights
Bacterial resistance to antibiotics is a major threat for human health
We have previously observed that peptides initially designed to bind Escherichia coli SC (EcSC) with high affinities poorly interact with Mycobacterium tuberculosis sliding clamp (MtSC) and BsSC, the binding pockets of all sliding clamp (SC) are structurally highly homologous[18]
In order to decipher the molecular basis of these different binding processes, we determined the thermodynamic parameters of the interaction between a reference ligand, namely peptide P7 (SI.[2] and SI.10) and different variants of EcSC, MtSC and BsSC (Table 1)
Summary
Bacterial resistance to antibiotics is a major threat for human health. According to several official reports, a return to the pre-antibiotic era during the 21st century is a realistic possibility[1]. In order to tackle this challenge, one strategy aims at identifying new bacterial molecular targets and at developing efficient molecules that will block their physiological functions. The bacterial processivity factor, referred to as the sliding clamp (SC), has been previously identified as a potential drugable new target 2 3 4 5 6. The ultimate proof of concept was recently brought by the natural cyclic peptides griselimycins that bind to Mycobacterium tuberculosis sliding clamp (MtSC) and display good in vivo anti-bacterial activity in animal models[7]. The molecular mechanisms that govern the SC/ligand interaction are not well understood4589
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