Mechanistic studies of the complex regulation of the signaling histidine kinase GacS from Pseudomonas aeruginosa

Structural and biochemical insights into the signaling mechanism of the histidine kinase GacS from Pseudomonas aeruginosa Project Summary The nosocomial pathogen Pseudomonas aeruginosa causes acute and persistent, chronic infections using distinct virulence mechanisms. The sensor signaling kinase GacS is a major player in regulating these virulence mechanisms. GacS itself is regulated through a complex set of non-canonical interactions. In recent work, our structural and biochemical studies have revealed that RetS and GacS form a novel heterodimeric DHp-DHp domain interface but that the overall GacS dimer remains intact. Consequently, GacS is predicted to undergo substantial conformational changes to accommodate RetS. Here, we propose to test a novel hypothesis positing that binding of the extracellular GacS ligand modulates RetS-GacS interactions by controlling the rigidity of the protein. Under the first aim, we propose experimentally validate the GacS kinking motion predicted by molecular dynamics simulations. The second part of the aim will directly measure the impact of ligand binding on shape and flexibility of a CitAGacS chimeric protein to test our hypothesis. Under aim 2 we seek to determine the identity of the elusive GacS ligand and use virtual screening to identify drug-like modulators of GacS. The in vitro screening approach is informed by our recent discovery of an inside-out signaling mechanism, whereby RetS primes GacS for signal recognition. Virtual screening is aided by the creation of a molecular model of the ligand-bound conformation of the sensory domain. Project Number: 1R21AI186059-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Florian Schubot | Institution: VIRGINIA POLYTECHNIC INST AND ST UNIV, BLACKSBURG, VA | Award Amount: $422,355 | Activity Code: R21 | Study Section: Macromolecular Structure and Function B Study Section[MSFB] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI18605901A1