DESCRIPTION (provided by applicant): The bacteria that inhabit the human intestinal tract are essential for immune and gastrointestinal development, pathogen protection, and complex carbohydrate digestion. Their ability to thrive in this niche is dependent upon their ability to extract carbohydrate nutrition from this highly competitive ecosystem. Bacteroidetes are numerically dominant Gram-negative members of the human gut microbiota that all rely upon similarly patterned outermembrane protein systems termed starch utilization (Sus)-like systems to capture carbohydrate nutrition. Every Sus-like system targets a unique glycan, and some species devote nearly 20% of their genomes towards encoding these proteins. Sus-like systems are only found in the Bacteroidetes, making these proteins attractive targets for manipulating the metabolism of these organisms to support human health. Our long-term goal is to understand the molecular events that support glycan utilization via Sus-like systems. In this proposal we will focus on the molecular interactions among the outermembrane proteins SusCDEFG in the prototypical starch utilization system (Sus) of Bacteroides thetaiotaomicron (Bt). We have determined the molecular structures of the starch-binding lipoproteins SusDEFG, but how these proteins interact with the TonB-dependent transporter SusC to facilitate glycan import is unknown. All Sus-like systems have homologs of SusC, and of SusD, and so understanding how SusCD interact with each other and with SusEFG will inform a general model of how Sus-like systems facilitate glycan uptake. In Specific Aim 1 we will determine the nature and stoichiometry of the SusCD interaction, as well as how SusEFG affect this assembly. Our working hypothesis is that SusD facilitates interactions between SusC and the SusEFG proteins. We will identify interacting Sus proteins via co-immunoprecipitation and proteomics. In parallel, we will examine the co- localization and stoichiometry of the Sus proteins in live cells y single molecule imaging. In Specific Aim 2 we will create a functional map of the SusC structure. Our working hypothesis is that the extracellular loops of SusC bind SusD. Although recombinant SusD has weak affinity for starch, the SusCD interaction may create a higher affinity site for maltooligosaccharides that enhances import. We will test targeted mutants of susC and susD in Bt and E. coli for their ability to interact with each other and starch. We will incorporate unnatual amino acids into SusC to introduce fluorescent tags and photo-crosslinkable residues that can map the interaction between SusC and SusD. Finally, we will determine the x-ray crystal structure of SusC with or without SusD to understand its topology and function. Together, these data will reveal the molecular details of the Sus complex assembly, and allow us to generate a working model of a conserved glycan acquisition paradigm that is unique to the Bacteroidetes. With these details...