Abstract GpsB is an intracellular anchor protein that interacts with a multitude of enzymes involved in cell division and cell wall synthesis. Comprehensive understanding of protein-protein interactions between GpsB and its partners will offer crucial information on the regulation of peptidoglycan synthesis and coordination of various cellular processes during cell growth and division, while providing a valuable target for novel antibiotic development. This proposal focuses on Staphylococcus aureus, a human pathogen whose GpsB is essential for its survival and contains unique structural features not observed in other homologs. The goal is to establish a multi-disciplinary platform to characterize GpsB interactions with potential partner proteins (PBP2, FtsZ, TarG), and to identify cyclic peptide and small molecule ligands for studying GpsB function and inhibition. The results will lay the foundation for a future in-depth analysis of the GpsB-dependent protein complexes underlying bacterial cell division, and an antibiotic discovery program to uncover cell-active high affinity compounds binding to GpsB.