One mechanism of resistance of S. aureus is the presence of a gene that encodes for the penicillin-binding protein PBP2a. PBP2a is responsible for the biosynthesis of bacterial cell wall by catalyzing a transpeptidation reaction in its active site. In contrast to other PBPs, the transpeptidase activity of which can be inhibited by beta-lactam antibiotics by the irreversible acylation of the catalytic serine, PBP2a does not experience modification by beta-lactam antibiotics readily. Therefore, the presence of PBP2a is a key factor in resistance in S. aureus, as it is able to catalyze cell-wall biosynthesis despite the challenge by beta-lactam antibiotics. The crystal structure of PBP2a has been determined, identifying two mobile loops that cover the active site in a closed conformation and an allosteric site 60 � away. Upon binding of substrate in the allosteric site, signal propagation takes place to cause the active site to open by movement of these two loops, allowing access for the transpeptidation reaction and ultimate biosynthesis of cell wall. In order to probe the conformational change around the active site, we have mutated an amino acid on each loop to a cysteine residue and labeled with the environmentally sensitive fluorescent coumarin probe, MDCC. Monitoring of the fluorescence of this labeled protein in the absence and presence of several antibiotics provides a convenient assay to probe the conformational changes. We have also introduced mutations within the allosteric site to determine the effect of allosteric substrate binding on signal propagation and active-site accessibility.

Fluorescent labeling of <i>Staphylococcus aureus</i> PBP2a to probe active-site conformational changes

246th National Meeting (2013)

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Fluorescent labeling of Staphylococcus aureus PBP2a to probe active-site conformational changes