Bacterial glycoconjugates are a diverse group of macromolecules that play a key role in bacterial survival and pathogenicity by mediating bacteria-host interactions. Despite their structural diversity, many glycoconjugates are made by prokaryotes utilizing a common mechanism, which includes transferring glycans to a Pren-PP- linked carrier at the membrane interface. The striking diversity of glycoconjugates is achieved through sequential addition of glycans by glycosyltransferases (GTs), which transfer sugars from soluble nucleotide-activated donors (NDP-sugars) to the lipid-based carrier molecule. Despite being ubiquitous across all kingdoms of life, GTs still represent an excellent target for antibiotics, due to the remarkable degree of selectivity they exhibit for structurally very similar sugars. Understanding the structural features that influence GT substrate selectivity and protein-protein interactions in pathogenic bacteria like Campylobacter species is critical for antibiotic development, and this research proposal strives to address that knowledge gap. To date, the substrates for C. jejuni GTs have been confirmed in the Campylobacter genus, but structural information for PglA, PglJ, PglH1, and PglH2 of C. concisus, another clinically significant human pathogen, is still unavailable. Additionally, characterizing GTs from the same organism's glycoconjugate biosynthetic pathway will offer the structural information needed for future protein-protein interaction studies and will pave the way for analysis to clarify how these proteins interact to modulate pathway flux. Aim 1 of this proposal will identify preferred GT substrates using nano-differential scanning fluorimetry (nanoDSF) and determine steady-state kinetic parameters using luminescence-based GT activity assays. Aim 2 will be to discover the detergent and buffer conditions required for crystallization of purified GTs, as well as to optimize protein crystals and collect X-ray crystal data to obtain high-resolution crystal structures. Aim 3 will be to characterize a number of orthologs from a GT-B sequence similarity network (SSN) in order to gain insight into GT evolution and to pinpoint the primary sequence variables that account for their various substrate specificities. Interprotein covariance between GT residues will be investigated via the GREMLIN method in order to identify interactions responsible for the hypothesized formation of supramolecular complexes between these enzymes. The resulting findings will reveal binding determinants controlling specificity for glycans prevalent in pathogenic prokaryotes and demonstrate crucial structure-function links and protein-protein interactions.