Mapping the pathway of membrane ?-barrel protein folding by the Bam complex. The rise of antibiotic resistance is a growing human health concern, but the development of new antibiotics against these pathogens, particularly Gram-negative bacteria, has not kept pace. A new class of antibiotics against Gram-negatives has not been identified in over 50 years, partly due to failures to identify new targets for inhibitor development. This proposal aims to evaluate a new potential target, the essential Bam complex, which folds and inserts ?-barrel proteins into the outer membrane (OM) of Gram-negative bacteria and is con- served from prokaryotes to eukaryotes. The OM is a defining characteristic of Gram-negatives and defects lead to increased membrane permeability or loss of cell viability. Within this membrane exist integral membrane proteins that exclusively adopt ?-barrel structures. The mechanism of their assembly remains unclear partially because of difficulties in obtaining high- resolution structural details of transient intermediate states during the assembly of these complex membrane proteins. To study this process, chemical and photocrosslinking strategies will be employed to capture and characterize folding intermediates on the Bam complex using the slow folding essential outer membrane protein, LptD. In this proposal, I have further slowed the assembly of LptD to trap intermediates in the process of folding on the Bam complex. I show that the entire tertiary structure of the LptD barrel is formed around its lipoprotein plug, LptE, prior to barrel closure and release from the Bam complex. Using this system as a model for protein folding, I will explore how Bam subunits coordinate with each other and with their substrates during barrel assembly by mapping the interaction surfaces and recognition motifs required for ?-strand insertion using an in vivo photocrosslinking strategy. I expect to identify residues in both essential components of the Bam complex, BamA and BamD, which are critical in facilitating barrel folding and insertion. Results will be recapitulated in a reconstitution of Bam complex activity in vitro. Lastly, I will evaluate if inhibition of such interactions by peptides containing these substrate sequence motifs is a viable strategy to target Gram-negatives. To achieve the outlined goals above, I propose the following Specific Aims:
Specific Aim I : To characterize the interaction surfaces between the Bam complex and a ?-barrel substrate in vivo Specific Aim II: To establish the requirements for substrate binding to th Bam complex and use these requirements to inhibit LptD/E assembly
The outer cell membrane is an important physical barrier that protects Gram-negative bacteria from the effects of many of our current clinical antibiotics. By studying how the machines that build the outer membrane operate, we hope to develop new strategies to interfere with their activity as a therapeutic option against Gram-negative pathogens.
|Mandler, Michael D; Baidin, Vadim; Lee, James et al. (2018) Novobiocin Enhances Polymyxin Activity by Stimulating Lipopolysaccharide Transport. J Am Chem Soc 140:6749-6753|
|Lee, James; Sutterlin, Holly A; Wzorek, Joseph S et al. (2018) Substrate binding to BamD triggers a conformational change in BamA to control membrane insertion. Proc Natl Acad Sci U S A 115:2359-2364|
|Wzorek, Joseph S; Lee, James; Tomasek, David et al. (2017) Membrane integration of an essential ?-barrel protein prerequires burial of an extracellular loop. Proc Natl Acad Sci U S A 114:2598-2603|
|Lee, James; Xue, Mingyu; Wzorek, Joseph S et al. (2016) Characterization of a stalled complex on the ?-barrel assembly machine. Proc Natl Acad Sci U S A 113:8717-22|