Ligand-induced allostery in the interaction of the Pseudomonas aeruginosa heme binding protein with heme oxygenase
JournalProceedings of the National Academy of Sciences of the United States of America
PublisherNational Academy of Sciences
MetadataShow full item record
AbstractA heme-dependent conformational rearrangement of the C-terminal domain of heme binding protein (PhuS) is required for interaction with the iron-regulated heme oxygenase (HemO). Herein, we further investigate the underlying mechanism of this conformational rearrangement and its implications for heme transfer via site-directed mutagenesis, resonance Raman (RR), hydrogen–deuterium exchange MS (HDX-MS) methods, and molecular dynamics (MD). HDX-MS revealed that the apo-PhuS C-terminal α6/α7/α8-helices are largely unstructured, whereas the apo-PhuS H212R variant showed an increase in structure within these regions. The increased rate of heme association with apo-PhuS H212R compared with the WT and lack of a detectable five-coordinate high-spin (5cHS) heme intermediate are consistent with a more folded and less dynamic C-terminal domain. HDX-MS and MD of holo-PhuS indicate an overall reduction in molecular flexibility throughout the protein, with significant structural rearrangement and protection of the heme binding pocket. We observed slow cooperative unfolding/folding events within the C-terminal helices of holo-PhuS and the N-terminal α1/α2-helices that are dampened or eliminated in the holo-PhuS H212R variant. Chemical cross-linking and MALDI-TOF MS mapped these same regions to the PhuS:HemO protein–protein interface. We previously proposed that the protein–protein interaction induces conformational rearrangement, promoting a ligand switch from His-209 to His-212 and triggering heme release to HemO. The reduced conformational freedom of holo-PhuS H212R combined with the increase in entropy and decrease in heme transfer on interaction with HemO further support this model. This study provides significant insight into the role of protein dynamics in heme binding and release in bacterial heme transport proteins.
Identifier to cite or link to this itemhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85016424644&doi=10.1073%2fpnas.1606931114&partnerID=40&md5=7f36307e69f97b876d593aabb0b38bef; http://hdl.handle.net/10713/9964
- Induced fit on heme binding to the Pseudomonas aeruginosa cytoplasmic protein (PhuS) drives interaction with heme oxygenase (HemO).
- Authors: O'Neill MJ, Bhakta MN, Fleming KG, Wilks A
- Issue date: 2012 Apr 10
- The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the delta-regioselective heme oxygenase of Pseudomonas aeruginosa.
- Authors: Bhakta MN, Wilks A
- Issue date: 2006 Sep 26
- The P. aeruginosa heme binding protein PhuS is a heme oxygenase titratable regulator of heme uptake.
- Authors: O'Neill MJ, Wilks A
- Issue date: 2013 Aug 16
- Identification of two heme-binding sites in the cytoplasmic heme-trafficking protein PhuS from Pseudomonas aeruginosa and their relevance to function.
- Authors: Block DR, Lukat-Rodgers GS, Rodgers KR, Wilks A, Bhakta MN, Lansky IB
- Issue date: 2007 Dec 18
- The role of the cytoplasmic heme-binding protein (PhuS) of Pseudomonas aeruginosa in intracellular heme trafficking and iron homeostasis.
- Authors: Kaur AP, Lansky IB, Wilks A
- Issue date: 2009 Jan 2