Biochemical and Crystallographic Studies of the Escherichia Coli β-Barrel Assembly Machine

Bacteria have developed resistance mechanisms to every class of antibiotics that has been created and combating the rise of multidrug-resistant bacteria—a major threat to public health—requires the continual development of novel antibacterial agents. Gram-negative bacteria are particularly difficult to target due to their dual-membrane cell envelope and highly impermeable outer membrane. Essential proteins at or near the cell surface of bacteria are therefore potential targets for novel antibiotic development. We sought to probe the mechanisms and structural details of two such essential bacterial proteins: the β-barrel assembly machine (Bam) of Escherichia coli and penicillin-binding protein 2 (PBP2) of Staphylococcus aureus.\n\nBam is a highly conserved five-protein complex that is situated in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. In E. coli the Bam complex performs the essential function of folding and inserting transmembrane β-barrel proteins into the cell’s outer membrane. In Chapter 2, biochemical assays aimed at elucidating the process by which outer membrane proteins are delivered to Bam are described. We reconstituted the Bam complex and subcomplexes in vitro to examine the role of chaperone proteins in delivering unfolded outer membrane proteins to Bam. We demonstrated that two β-barrel protein substrates with large soluble periplasmic domains have the ability to fold on the complex independent of a chaperone.\n\nIn vitro biochemical experiments with the Bam complex cannot provide atomic-level mechanistic insight into how the complex binds, folds, and inserts transmembrane β-barrels into the outer membrane. In Chapter 3, efforts toward obtaining a crystal structure of Bam are described. Structures of the five Bam proteins had been solved individually, but the structure of the Bam complex or a Bam subcomplex had not been reported. We successfully crystallized Bam subcomplexes and made progress toward obtaining a crystal structure of this macromolecular machine.\n\nIn Chapter 4, we report the use of a fluorescence displacement assay to identity putative small molecule inhibitors of the S. aureus PBP2 protein. PBP2 catalyzes the essential transglycosylation and transpeptidation steps in the synthesis of the bacterial cell wall and is therefore an attractive target for novel antibiotics. We report progress toward the cocrystallization of PBP2 with inhibitors identified using the fluorescence displacement assay.