Project Summary/Abstract The rise of drug resistant bacteria is a rapidly evolving threat to human health. Pathogenic bacteria have developed several mechanisms to battle the threat posed by antimicrobial compounds and survive in niche environments within the human body. The overarching goal in our laboratory is to understand at a molecular level how pathogenic bacteria utilize specific membrane protein complexes to meet these specialized needs. We place a particular emphasis on understanding the structure and function of membrane transporters that move molecules and signals across bacterial membranes, and protein complexes that allow bacteria to sense and respond to environmental stimuli. In order to achieve these goals, we routinely combine high-resolution cryo- electron microscopy with biochemical and computational methods to gain insight into the structure, conformational dynamics, and overall function of membrane transporters and signaling complexes. Our primary focus during the award period will be to understand how Gram-positive species use dedicated membrane protein machinery to sense and evade attack by antimicrobial peptides. Antimicrobial peptides such as vancomycin are some of the most powerful antibiotics currently in clinical use and are considered a treatment option of last resort. However, infection with Gram-positive organisms such as vancomycin-resistant Enterococcus or Staphylococcus continue to threaten healthcare settings, and leave infected patients with limited treatment options. Many Gram-positive species express membrane protein complexes known as “Bce modules” (BCEMs) that contain an ABC transporter and a two-component system that work in tandem to sense and respond to attack by antimicrobial peptides. Our primary goals are to obtain a complete understanding of how the ABC transporter component of BCEMs recognizes and provides resistance to antimicrobial peptides, and how conformational cycling of the ABC transporter initiates signaling through the two-component system via a flux-sensing mechanism. A comprehensive study of the structure, conformational dynamics, kinetic mechanisms, and in vivo activity of BCEMs will be performed in order to understand how the constituents of these modules work in synergy to sense and respond to antimicrobial peptides. At the culmination of our studies we will have established a structure-driven understanding of the membrane protein complexes that allow Gram-positive pathogens to sense and respond to different antimicrobial peptides. Our long-term vision is to build a comprehensive model of the different protein machineries used by microbial pathogens to circumvent our most powerful antibiotics. Detailed structural and functional analysis of these protein complexes will set the stage for development of new and improved antimicrobial compounds and targeted therapies for drug resistant microbial infections.