PAS domain of flagellar histidine kinase FlrB has a unique architecture and binds heme as a sensory ligand in an unconventional fashion.

Phosphorylation of the σ54-dependent transcription activator FlrC by the sensor histidine kinase FlrB is essential for flagellar synthesis of Vibrio cholerae. Despite that, the structure, sensory signal, and mechanistic basis of function of FlrB were elusive. Here, we report the crystal structure of the sensory PAS domain of FlrB in its functional dimeric state that exhibits a unique architecture. Series of biochemical/biophysical experiments on different constructs and mutants established that heme binds hydrophobically as sensory ligand in the shallow ligand-binding cleft of FlrB-PAS without axial coordination. Intriguingly, ATP binding to the C-terminal ATP-binding (CA) domain assists PAS domain to bind heme, vis-à-vis, heme binding to the PAS facilitates ATP binding to the CA domain. We hypothesize that synergistic binding of heme and ATP triggers conformational signaling in FlrB, leading to downstream flagellar gene transcription. Enhanced swimming motility of V. cholerae with increased heme uptake supports this proposition.

Mechanistic insights of ABC importer HutCD involved in heme internalization by Vibrio cholerae.

Heme internalization by pathogenic bacteria inside a human host to accomplish the requirement of iron for important cellular processes is of paramount importance. Despite this, the mechanism of heme import by the ATP-binding-cassette (ABC) transporter HutCD in Vibrio cholerae remains unexplored. We have performed biochemical studies on ATPase HutD and its mutants, along with molecular modelling, docking and unbiased all-atom MD simulations on lipid-solvated models of permease-ATPase complex HutCD. The results demonstrated mechanisms of ATP binding/hydrolysis and trapped transient and global conformational changes in HutCD, necessary for heme internalization. ATPase HutD forms a dimer, independent of the permease HutC. Each HutD monomer canonically binds ATP in a 1:1 stoichiometry. MD simulations demonstrated that a rotational motion of HutC dimer occurs synchronously with the inter-dimeric D-loop interactions of HutDs. F151 of TM4-TM5 loop of HutC, packs with ATP and Y15 of HutD, initiating 'cytoplasmic gate opening' which mimics an 'outward-facing' to 'inward-facing' conformational switching upon ATP hydrolysis. The simulation on 'inward-facing' HutCD culminates to an 'occluded' state. The simulation on heme-docked HutCD indicated that the event of heme release occurs in ATP-free 'inward-facing' state. Gradual conformational changes of the TM5 helices of HutC towards the 'occluded' state facilitate ejection of heme.