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

Daniel J. Deredge, Weiliang Huang, Colleen Hui, Hirotoshi Matsumura, Zhi Yue, Pierre Moenne-Loccoz, Jana Shen, Patrick L. Wintrode, Angela Wilks

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 highspin (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.

Original languageEnglish (US)
Pages (from-to)3421-3426
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number13
DOIs
StatePublished - Mar 28 2017

Fingerprint

Heme Oxygenase (Decyclizing)
Heme
Pseudomonas aeruginosa
Ligands
Deuterium
Hydrogen
Proteins
Molecular Dynamics Simulation
Apolipoproteins C
Hemeproteins
heme-binding protein
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Entropy
Site-Directed Mutagenesis
Carrier Proteins
Iron

Keywords

  • Allostery
  • Heme Binding
  • Heme Transfer
  • Protein-Protein Interaction
  • Pseudomonas Aeruginosa

ASJC Scopus subject areas

  • General

Cite this

Ligand-induced allostery in the interaction of the Pseudomonas aeruginosa heme binding protein with heme oxygenase. / Deredge, Daniel J.; Huang, Weiliang; Hui, Colleen; Matsumura, Hirotoshi; Yue, Zhi; Moenne-Loccoz, Pierre; Shen, Jana; Wintrode, Patrick L.; Wilks, Angela.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 114, No. 13, 28.03.2017, p. 3421-3426.

Research output: Contribution to journalArticle

Deredge, Daniel J. ; Huang, Weiliang ; Hui, Colleen ; Matsumura, Hirotoshi ; Yue, Zhi ; Moenne-Loccoz, Pierre ; Shen, Jana ; Wintrode, Patrick L. ; Wilks, Angela. / Ligand-induced allostery in the interaction of the Pseudomonas aeruginosa heme binding protein with heme oxygenase. In: Proceedings of the National Academy of Sciences of the United States of America. 2017 ; Vol. 114, No. 13. pp. 3421-3426.
@article{6c6b2c6489f6449391429017720dfd6d,
title = "Ligand-induced allostery in the interaction of the Pseudomonas aeruginosa heme 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 highspin (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.",
keywords = "Allostery, Heme Binding, Heme Transfer, Protein-Protein Interaction, Pseudomonas Aeruginosa",
author = "Deredge, {Daniel J.} and Weiliang Huang and Colleen Hui and Hirotoshi Matsumura and Zhi Yue and Pierre Moenne-Loccoz and Jana Shen and Wintrode, {Patrick L.} and Angela Wilks",
year = "2017",
month = "3",
day = "28",
doi = "10.1073/pnas.1606931114/-/DCSupplemental",
language = "English (US)",
volume = "114",
pages = "3421--3426",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "13",

}

TY - JOUR

T1 - Ligand-induced allostery in the interaction of the Pseudomonas aeruginosa heme binding protein with heme oxygenase

AU - Deredge, Daniel J.

AU - Huang, Weiliang

AU - Hui, Colleen

AU - Matsumura, Hirotoshi

AU - Yue, Zhi

AU - Moenne-Loccoz, Pierre

AU - Shen, Jana

AU - Wintrode, Patrick L.

AU - Wilks, Angela

PY - 2017/3/28

Y1 - 2017/3/28

N2 - 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 highspin (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.

AB - 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 highspin (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.

KW - Allostery

KW - Heme Binding

KW - Heme Transfer

KW - Protein-Protein Interaction

KW - Pseudomonas Aeruginosa

UR - http://www.scopus.com/inward/record.url?scp=85016424644&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85016424644&partnerID=8YFLogxK

U2 - 10.1073/pnas.1606931114/-/DCSupplemental

DO - 10.1073/pnas.1606931114/-/DCSupplemental

M3 - Article

C2 - 28289188

AN - SCOPUS:85016424644

VL - 114

SP - 3421

EP - 3426

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 13

ER -