Copurification of the FpvA ferric pyoverdin receptor of Pseudomonas aeruginosa with its iron-free ligand: implications for siderophore-mediated iron transport.
The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer membrane transport protein that catalyzes uptake of ferric pyoverdin across the outer membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA-deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric-PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA-PaA-Fe complex in the presence of PaA-Fe or citrate-Fe. The circular dichroism spectrum of FpvA indicated a 57% beta-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. This truncated form still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N-terminal periplasmic extensions of FecA from Escherichia coli and PupB from Pseudomonas putida, which were previously shown to be involved in signal transduction. This suggests a similar function for FpvA. The mechanism of iron transport in P. aeruginosa via the pyoverdin pathway is discussed in the light of all these new findings.
Département des Récepteurs et Protéines Membranaires, UPR 9050 CNRS, ESBS, Strasbourg, France. schalk@esbs.u-strasbg.fr
I J
IJ
Schalk
P
P
Kyslik
D
D
Prome
A
A
van Dorsselaer
K
K
Poole
M A
MA
Abdallah
F
F
Pattus
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D001692:Biological Transport; D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism; D006868:Hydrolysis; D007502:Iron Chelating Agents / Q000302:isolation & purification / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010860:Pigments, Biological / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D017262:Siderophores / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C082326:FpvA protein, Pseudomonas aeruginosa; D007502:Iron Chelating Agents; D008024:Ligands; D009842:Oligopeptides; D010860:Pigments, Biological; D017262:Siderophores; C042453:pyoverdin; D010450:Endopeptidases