Structural Characterization of the Hemophore HasAp from Pseudomonas aeruginosa: NMR Spectroscopy Reveals Protein−Protein Interactions between Holo-HasAp and Hemoglobin^,

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 met-hemoglobin 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 15 Nlabeled 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 15 N-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, † This work was supported by grants from the National Institute of Health, GM-50503 (M.R.), NSF-MCB-0818488 (M.R.) and NSF-MCB-0811888 (P.M.L.). ‡ Coordinates and crystallographic structure factors for HasAp have been deposited in the protein data bank under accession code 3ELL.Backbone resonance assignments for truncated and full-length holo-HasAp have been deposited in the BMRB under access code 15962 and 15963, respectively. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 January 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptsuggesting that the disordered tail in the full-length HasAp must be proteolyzed in the extracellular milieu to make HasAp a more efficient hemophore.The preferred aerobic metabolism of Pseudomonas aeruginosa requires respiratory enzymes that need iron or iron-containing cofactors for their function. The extremely low concentrations of free iron in mammalian hosts trigger a stress response in the opportunistic P. aeruginosa (and in many other pathogens) that involves the deployment of several iron-acquisition systems (1-4). The systems involved in the capture of iron typically fall in two categories, (i) excretion of low-molecular weight i...

Structural flexibility of the heme cavity in the cold‐adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125

“…This is confirmed by the corresponding high-frequency RR spectrum ( for ferric Ph-2/2HbO at pH 7.6 [13] where, probably due to different preparation procedures, multiple LS forms were observed (see Materials and methods). However, as reported in that case, the absorption maxima of the LS form are quite unusual, and reminiscent of those of ferric Chlamydomonas chloroplast Hb [23] and of the hemophore HasA proteins from Serratia marcescens and Pseudomonas aeruginosa [24,25]. Nevertheless, they are very different from either a LS His-Fe-His (that exhibits well defined absorption bands at about 535 and 565 nm) or a His-Fe-OH heme complex as observed at alkaline pH (see below) [26].…”

Targeting iron uptake to controlPseudomonas aeruginosainfections in cystic fibrosis

“…Two haem uptake systems have been described in P. aeruginosa (Phu and Has) [49]. The Phu system relies on direct binding of haem or haem-containing proteins to a membrane-bound receptor, whereas the Has system secretes a haem-binding protein (HasAp) which is reabsorbed through the Has receptor (HasR) when bound to haem [50,51]. The P. aeruginosa genome contains a third haem receptor-encoding gene (hxuC); however, its functional regulation has yet to be characterised [52].…”

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

“…The wavelength maxima suggest the presence of various species, namely a His-Fe-H 2 O six-coordinate high-spin (6cHS) form [bands at 503 and charge-transfer transition (CT1) at 635 nm] and at least one 6c-low-spin (LS) heme (bands at 533 and 570 nm). The absorption maxima of the LS forms are quite unusual, reminiscent of those of ferric Chlamydomonas Hb [20] and the hemophore HasA proteins from Serratia marcescens and Pseudomonas aeruginosa [21,22], and they are very different from either a LS His-Fe-His (that exhibits well-defined absorption bands at about 535 and 565 nm) or a His-Fe-OH heme complex (that exhibits well-defined absorption bands at about 540 and 580 nm) [23]. Therefore, on the basis of the similarity with the UV-vis spectrum of ferric Chlamydomonas Hb and HasA hemophores, a His and a Tyr ligand are suggested to occupy the fifth and sixth coordination positions, respectively.…”