This proposal focuses on the structural and functional characterization of antimicrobial natural products. The precise molecular details of how these agents fold and how they recognize their targets will be derived from high resolution X-ray crystal structures of drugs and drug:target complexes. Three classes of compounds will be studied. The first class includes vancomycin and related glycopeptide antibiotics. Structural experiments will test hypotheses about the conformational changes accompanying ligand recognition and the role of higher order structural assemblies in peptidoglycan recognition by vancomycin. Structures will also be determined for Group III (teicoplanin-like) antibiotics, including the newly introduced therapeutic agent dalbavancin, allowing for a comparison of Group I (vancomycin-like) and Group III antibiotics, and illuminating how ligand recognition and oligomerization behavior differ between the two groups. Novel glycopeptide compounds have been designed based upon existing structural data, and will be synthesized and characterized. The second class of bacteriocidal natural products to be studied are molecules that recognize polyprenyl pyrophosphate-containing ligands on bacterial cells, such as the Lipid II intermediate in peptidoglycan biosynthesis. Crystallographic studies will be used to reveal the determinants of molecular recognition for these different agents, in an effort to establish their mechanisms of action and informing the future design of related therapeutic agents. The specific molecules under study include ramoplanin, enduracidin, and bacitracin. The third class of antibiotics comprises cyclic decapeptides that permeabilize bacterial membranes. While these are effective antimicrobials, they can also be hemolytic;therefore crystallography will be used to study the structures of an array of compounds with differing bacteriocidal and hemolytic activities. Comparison of these will identify the structural determinants that control the desired antibacterial activity and the unwanted side effects, and thus aid in the development of improved agents. Relevance to public health: The prevalence of resistant bacterial pathogens is rendering current antibiotics ineffective, and making it essential that new drugs be developed to fight infection. This project will provide essential information about naturally occurring antimicrobial compounds. This information will facilitate the rational development of these natural compounds into next generation therapeutic agents.