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    Ligand-induced allostery in the interaction of the Pseudomonas aeruginosa heme binding protein with heme oxygenase

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    Author
    Deredge, D.J.
    Huang, W.
    Hui, C.
    Date
    2017
    Journal
    Proceedings of the National Academy of Sciences of the United States of America
    Publisher
    National Academy of Sciences
    Type
    Article
    
    Metadata
    Show full item record
    See at
    https://www.doi.org/10.1073/pnas.1606931114
    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.
    Keyword
    Allostery
    Heme Binding
    Heme Transfer
    Protein-Protein Interaction
    Pseudomonas Aeruginosa
    Identifier to cite or link to this item
    https://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
    ae974a485f413a2113503eed53cd6c53
    10.1073/pnas.1606931114
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    UMB Open Access Articles 2017

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