Molecular Insights into the Conformational Dynamics of the Novel Dispersion Sensor Histidine Kinase DspS.

Dispersion sensor (DspS) is a membrane-bound histidine kinase in Pseudomonas aeruginosa that senses cis-2-decenoic acid (CDA), a fatty acid signal regulating biofilm dispersion, motility, and virulence. However, its structural mechanism of signal transduction remains poorly defined. Here, we combined AlphaFold-Multimer modeling, multiple-replica accelerated molecular dynamics simulations, and Markov state modeling (MSM) to investigate the conformational dynamics of full-length dimeric DspS. Simulations focused on the cyclases/histidine kinase-associated sensory extracellular domain and transmembrane (TM) helices under four conditions: apo, dual-CDA-bound, dual arginine-to-alanine substitution, and single-CDA-bound. The apo form displayed a loosely packed TM2-TM2' helical arrangement, whereas dual CDA binding promoted a tightly crossed, signaling-competent configuration. In contrast, disruption of the conserved Arg residue weakened ligand anchoring and partially destabilized this packing. Under single-CDA binding, the DspS dimer adopted a primed state arising from asymmetrical ligand occupancy. In this state, the dimer bent toward the membrane interface, suggesting a distinct structural rearrangement relative to the apo and dual-bound forms. Such asymmetry may represent a mechanistic adaptation to fluctuating CDA levels, allowing graded receptor activation under dynamic environmental conditions. MSM analysis further revealed conformational plasticity, showing that full ligand occupancy stabilizes a network of metastable states and enables transitions within an active-like conformational ensemble. These findings provide the first atomistic view of DspS conformational switching, establishing a structural framework to study related histidine kinases and design antivirulence strategies targeting DspS-mediated biofilm control.