Heme binding to the IsdE(M78A; H229A) double mutant: challenging unidirectional heme transfer in the iron-regulated surface determinant protein heme transfer pathway of Staphylococcus aureus.
The pathogenic bacterium Staphylococcus aureus has adopted specialized mechanisms for scavenging iron from its host. The cell-wall- and cell-membrane-associated iron-regulated surface determinant (Isd) proteins (IsdH, IsdB, IsdA, IsdC, IsdDEF, IsdG, and IsdI) allow S. aureus to scavenge iron from the heme in hemoglobin and haptoglobin-hemoglobin. Of these, IsdE chaperones heme to the ATP-binding-cassette-type transmembrane transporter (IsdF). IsdH, IsdB, IsdA, and IsdC contain at least one heme-binding near transporter (NEAT) domain. Previous studies have shown that ferric heme is transferred unidirectionally in the sequence IsdA-NEAT (Tyr-proximal amino acid) → IsdC-NEAT (Tyr) → IsdE (His). IsdA-NEAT does not transfer heme directly to IsdE. To challenge and probe this unusual unidirectional mechanism, the double mutant IsdE(M78A; H229A)-IsdE(MH)-was constructed and used in studies of heme transfer between IsdA-NEAT, IsdC-NEAT, and IsdE. This study probed the specific requirements in the heme binding site that enforce the unidirectional property of the system. Significantly, heme transfer from holo-IsdE(MH) to apo-IsdA-NEAT now occurs, breaking the established mechanism. The unique unidirectional heme-transfer properties now function under an affinity-driven mechanism. Overall, the heme proximal and distal ligands must play a crucial role controlling a gate that stops heme transfer between the native IsdE and IsdA-NEAT. We propose that these amino acids are the key control elements in the specific unidirectional protein-protein-gated release mechanism exhibited by the Isd system.
Department of Chemistry, The University of Western Ontario, London, ON, N6A 5B7, Canada.
Michael T
MT
Tiedemann
Martin J
MJ
Stillman
D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003198:Computer Simulation; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D013211:Staphylococcus aureus / Q000737:chemistry / Q000378:metabolism
D000942:Antigens, Bacterial; D002352:Carrier Proteins; C488728:IsdE protein, Staphylococcus aureus; D006418:Heme; D007501:Iron; D005982:Glutathione Transferase