Abstract
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.