title: Ligand-induced allostery in the interaction of thePseudomonas aeruginosaheme binding protein with heme oxygenase.
abstract: A 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.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Deredge
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201.
authors.forename: Weiliang
authors.initials: W
authors.lastname: Huang
authors.affiliation: Institute of Environmental Health, Division of Environmental and Biomolecular Systems, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Colleen
authors.initials: C
authors.lastname: Hui
authors.affiliation: Institute of Environmental Health, Division of Environmental and Biomolecular Systems, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Matsumura
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Yue
authors.affiliation: Institute of Environmental Health, Division of Environmental and Biomolecular Systems, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Pierre
authors.initials: P
authors.lastname: Moënne-Loccoz
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201.
authors.forename: Jana
authors.identifier: 0000-0002-3234-0769
authors.initials: J
authors.lastname: Shen
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201.
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Wintrode
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201; awilks@rx.umaryland.edu.
authors.forename: Angela
authors.initials: A
authors.lastname: Wilks
mesh_terms: D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D008024:Ligands; D006419:Heme Oxygenase (Decyclizing)
keywords: Pseudomonas aeruginosa; allostery; heme binding; heme transfer; protein–protein interaction