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.
Schalk
I J
IJ
Département des Récepteurs et Protéines Membranaires, UPR 9050 CNRS, ESBS, Strasbourg, France. schalk@esbs.u-strasbg.fr
Kyslik
P
P
Prome
D
D
van Dorsselaer
A
A
Poole
K
K
Abdallah
M A
MA
Pattus
F
F
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