Virulence in Pseudomonas aeruginosa (PA) depends on complex regulatory networks, involving phosphorelay systems based on two-component systems (TCSs). The GacS/GacA TCS is a master regulator of biofilm formation, swarming motility, and virulence. GacS is a membrane-associated unorthodox histidine kinase (HK) whose phosphorelay signaling pathway is inhibited by the RetS hybrid HK. Here we provide structural and functional insights into the interaction of GacS with RetS. The structure of the GacS-HAMP-H1 cytoplasmic regions reveals an unusually elongated homodimer marked by a 135 Å long helical bundle formed by the HAMP, the signaling helix (S helix) and the DHp subdomain. The HAMP and S helix regions are essential for GacS signaling and contribute to the GacS/RetS binding interface. The structure of the GacS D1 domain together with the discovery of an unidentified functional ND domain, essential for GacS full autokinase activity, unveils signature motifs in GacS required for its atypical autokinase mechanism. [Display omitted] • GacS-HAMP-H1 crystal structure reveals a 135 Å long helical bundle • The atypical autokinase activity of GacS depends on a pseudoreceiver domain • The deletion of the GacS-HAMP region hyperactivates GacS signaling in vivo • The GacS-HAMP domain is involved in the formation of the GacS:RetS complex Fadel et al. study the structure and function of GacS histidine kinase from Pseudomonas aeruginosa. GacS plays a central role in regulating biofilm development and is inhibited by RetS through direct interactions. The GacS-HAMP region is involved in these interactions and regulates GacS activity as well as a pseudoreceiver domain. [ABSTRACT FROM AUTHOR]