Abstract:
Beta-lactamases hydrolyze the beta-lactam ring of antibiotics, rendering them ineffective. Understanding the inhibitor recognition mechanism of beta-lactamases will give important information toward the fight against beta-lactamase mediated antibiotic resistance. Most inhibitors of beta-lactamase bind to the active site and thus inhibit beta-lactamase in a competitive manner. In this study, inhibitor recognition mechanism of beta-lactamase was examined using molecular dynamics simulations and in vitro kinetic experiments. Simulations were performed on apo and BLIP bound forms of two clinically relevant beta-lactamases, TEM-1 and SHV-1 in order to elucidate the change in dynamics and energetics upon BLIP binding. An allosteric inhibitor binding site, near the H10 helix (residues 218-230), was previously discovered in TEM-1 and SHV-1. Multiple sequence alignment of beta-lactamases shows that Trp229, which resides on the H10 helix, is a highly conserved residue within the beta-lactamase family. Simulations were repeated on the W229A mutant forms of TEM-1 and SHV-1 beta-lactamase in order to investigate the role of Trp229 in ligand recognition mechanism of beta-lactamase. Simulations resulted in higher H10 mobility in TEM-1 and SHV-1 beta-lactamases in the presence of BLIP and higher cross correlations with BLIP. Higher affinity toward BLIP was observed in W229A mutant TEM-1 but not in mutant SHV-1 beta-lactamase. The results indicate the presence of the communication between allosteric site and active site of beta-lactamase. Molecular dynamics simulations were performed on 10 different TEM-1 – peptide complexes, with various mutations done on the peptide. The structural, dynamic and energetic examination of the simulation trajectories revealed that the G48F mutant of the peptide comprising BLIP residues 45 to 53 had strong nonbonded interactions between enzyme and peptide and higher affinity toward TEM-1. In vitro experiments were carried out with four different peptides. The peptide comprising BLIP residues 45 to 53 and a peptide that has additional pVEC residues at the N-terminus of its sequence designed based on 45-53 region of BLIP were found to have similar inhibitory effects on TEM-1 beta-lactamase activity.
