Structural characterization of the hemophore HasAp from pseudomonas aeruginosa

NMR Spectroscopy reveals protein-protein interactions between holo-HasAp and hemoglobin

Aileen Y. Alontaga, Juan Carlos Rodriguez, Ernst Schönbrunn, Andreas Becker, Todd Funke, Erik T. Yukl, Takahiro Hayashi, Jordan Stobaugh, Pierre Moenne-Loccoz, Mario Rivera

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Pseudomonas aeruginosa secretes a 205 residue long hemophore (full-length HasAp) that is subsequently cleaved at the C'-terminal domain to produce mainly a 184 residue long truncated HasAp that scavenges heme [Letoffé, S., Redeker, V., and Wandersman, C. (1998) Mol. Microbiol. 28, 1223-1234]. HasAp has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The X-ray crystal structure of truncated HasAp revealed a polypeptide αβ fold and a ferriheme coordinated axially by His32 and Tyr75, with the side chain of His83 poised to accept a hydrogen bond from the Tyr75 phenolic acid group. NMR investigations conducted with full-length HasAp showed that the carboxyl- terminal tail (21 residues) is disordered and conformationally flexible. NMR spectroscopic investigations aimed at studying a complex between apo-HasAp and human methemoglobin were stymied by the rapid heme capture by the hemophore. In an effort to circumvent this problem NMR spectroscopy was used to monitor the titration of 15N-labeled holo-HasAp with hemoglobin. These studies allowed identification of a specific area on the surface of truncated HasAp, encompassing the axial ligand His32 loop that serves as a transient site of interaction with hemoglobin. These findings are discussed in the context of a putative encounter complex between apo-HasAp and hemoglobin that leads to efficient hemoglobin-heme capture by the hemophore. Similar experiments conducted with full-length 15N-labeled HasAp and hemoglobin revealed a transient interaction site in full-length HasAp similar to that observed in the truncated hemophore. The spectral perturbations observed while investigating these interactions, however, are weaker than those observed for the interactions between hemoglobin and truncated HasAp, suggesting that the disordered tail in the full-length HasAp must be proteolyzed in the extracellular milieu to make HasAp a more efficient hemophore.

Original languageEnglish (US)
Pages (from-to)96-109
Number of pages14
JournalBiochemistry
Volume48
Issue number1
DOIs
StatePublished - Jan 13 2009

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Biomolecular Nuclear Magnetic Resonance
Pseudomonas aeruginosa
Nuclear magnetic resonance spectroscopy
Hemoglobins
Heme
Proteins
Truncated Hemoglobins
Tail
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Methemoglobin
X ray crystallography
X Ray Crystallography
Titration
Hydrogen
Hydrogen bonds
Crystal structure
X-Rays
Ligands
X rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural characterization of the hemophore HasAp from pseudomonas aeruginosa : NMR Spectroscopy reveals protein-protein interactions between holo-HasAp and hemoglobin. / Alontaga, Aileen Y.; Rodriguez, Juan Carlos; Schönbrunn, Ernst; Becker, Andreas; Funke, Todd; Yukl, Erik T.; Hayashi, Takahiro; Stobaugh, Jordan; Moenne-Loccoz, Pierre; Rivera, Mario.

In: Biochemistry, Vol. 48, No. 1, 13.01.2009, p. 96-109.

Research output: Contribution to journalArticle

Alontaga, Aileen Y. ; Rodriguez, Juan Carlos ; Schönbrunn, Ernst ; Becker, Andreas ; Funke, Todd ; Yukl, Erik T. ; Hayashi, Takahiro ; Stobaugh, Jordan ; Moenne-Loccoz, Pierre ; Rivera, Mario. / Structural characterization of the hemophore HasAp from pseudomonas aeruginosa : NMR Spectroscopy reveals protein-protein interactions between holo-HasAp and hemoglobin. In: Biochemistry. 2009 ; Vol. 48, No. 1. pp. 96-109.
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abstract = "Pseudomonas aeruginosa secretes a 205 residue long hemophore (full-length HasAp) that is subsequently cleaved at the C'-terminal domain to produce mainly a 184 residue long truncated HasAp that scavenges heme [Letoff{\'e}, S., Redeker, V., and Wandersman, C. (1998) Mol. Microbiol. 28, 1223-1234]. HasAp has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The X-ray crystal structure of truncated HasAp revealed a polypeptide αβ fold and a ferriheme coordinated axially by His32 and Tyr75, with the side chain of His83 poised to accept a hydrogen bond from the Tyr75 phenolic acid group. NMR investigations conducted with full-length HasAp showed that the carboxyl- terminal tail (21 residues) is disordered and conformationally flexible. NMR spectroscopic investigations aimed at studying a complex between apo-HasAp and human methemoglobin were stymied by the rapid heme capture by the hemophore. In an effort to circumvent this problem NMR spectroscopy was used to monitor the titration of 15N-labeled holo-HasAp with hemoglobin. These studies allowed identification of a specific area on the surface of truncated HasAp, encompassing the axial ligand His32 loop that serves as a transient site of interaction with hemoglobin. These findings are discussed in the context of a putative encounter complex between apo-HasAp and hemoglobin that leads to efficient hemoglobin-heme capture by the hemophore. Similar experiments conducted with full-length 15N-labeled HasAp and hemoglobin revealed a transient interaction site in full-length HasAp similar to that observed in the truncated hemophore. The spectral perturbations observed while investigating these interactions, however, are weaker than those observed for the interactions between hemoglobin and truncated HasAp, suggesting that the disordered tail in the full-length HasAp must be proteolyzed in the extracellular milieu to make HasAp a more efficient hemophore.",
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AU - Becker, Andreas

AU - Funke, Todd

AU - Yukl, Erik T.

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AU - Rivera, Mario

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