Structure and dynamics of penicillin-binding protein

Abstract

Bacteria maintain their cell shape and resist the cytosol's internal pressure by constructing a cross-linked polymer, peptidoglycan. The penicillin-binding proteins (PBPs) are a ubiquitous family of glycosyltransferase and transpeptidase enzymes that maintain and develop peptidoglycan in bacteria. The transpeptidase domain is the primary target of β-lactam antibiotics and is particularly interesting to drug discovery and the study of bacterial growth and division. Despite their ubiquity and interest, the individual roles of PBPs are not well understood, partly due to a lack of selective chemical tools. To this end, the Carlson lab in the Department of Chemistry at UMN has developed PBP1b and 2x co-selective β-lactone probes that are click-chemistry capable activity-based probes (ABPs). This thesis primarily focuses on dynamics-contextualized structure-activity relationships of β-lactones and β-lactam inhibitors to rationalize and predict affinity and selectivity for individual inhibitors in PBP1b and PBP2x. Chapters 2 and 3 describe structure and dynamics analyses of inhibitors bound to PBP1b, providing a greater context for binding modes within and between series of inhibitors. Importantly, we found the average distance of the β-lactam carboxylate to the motif III lysine to be capable of rank-ordering inhibitors by their kinact/KI. Additionally, we identified a chloride ion in both X-ray crystallography and molecular dynamics to be ubiquitous in non-carboxylate containing β-lactone inhibitors, potentially rationalizing their ability to bind despite lacking the negatively charged moiety. Chapter 4 focuses on using and optimizing free energy perturbation (FEP) simulations to predict affinity and selectivity changes for analogs of 7Az. This work expanded the available understanding of simulation parameters necessary for rigorous free energy estimates for irreversible covalent inhibitors. Finally, we propose multiple 7Az analogs predicted to have greater selectivity for PBP1b over PBP2x compared to currently available β-lactone ABPs.