The binding mechanism of pyoverdin with the outer membrane receptor FpvA in Pseudomonas aeruginosa is dependent on its iron-loaded status.
In iron-deficient conditions, Pseudomonas aeruginosa secretes a major fluorescent siderophore named pyoverdin (Pvd), which after chelating iron(III) is transported back into the cell via its outer membrane receptor FpvA. FpvA is a TonB-dependent transport protein and has the ability to bind Pvd in its apo- or iron-loaded form. The fluorescence properties of Pvd were used to determine the binding kinetics of metal-free and metal-loaded Pvd to FpvA and showed two major features. First, the kinetics of formation of the FpvA-Pvd complex, in vivo and in vitro, are markedly slower compared to those observed for FpvA-Pvd-metal. Second, apo-Pvd and Pvd-metal absorbed with biphasic kinetics to FpvA: the bimolecular step (association of the ligand with the receptor) is followed by a slower step (t(1/2) values of 5 and 34 min for Pvd-metal and Pvd, respectively) that presumably leads to a more stable complex. The most likely explanation for this second step is that the binding of the ligand to the receptor induces a conformational change on FpvA, which may be different, depending on the loading status of Pvd. Analysis of the dissociation of metal-free Pvd from FpvA revealed an energy and a TonB dependency. The dissociation of iron-free Pvd from FpvA in the absence of the TonB protein occurs with slow kinetics in the range of hours, but it can be highly activated by the protonmotive force and TonB to reach a kinetic with a t(1/2) of 1 min. Apparently, under iron-limited conditions, TonB activates the FpvA receptor, resulting in a fast release of iron-free Pvd and generating an unloaded FpvA receptor, competent for binding extracellular Pvd-Fe.
Clément
Emilie
E
Departement des Récepteurs et Protéines Membranaires, UPR 9050 CNRS, ESBS, Bld Sébastien Brandt, BP 10412, F-67413 Illkirch, Strasbourg, France.
Mesini
Philippe J
PJ
Pattus
Franc
F
Schalk
Isabelle J
IJ
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D009842:Oligopeptides; D010860:Pigments, Biological / Q000378:metabolism*; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001425:Bacterial Outer Membrane Proteins; C082326:FpvA protein, Pseudomonas aeruginosa; D008024:Ligands; D009842:Oligopeptides; D010860:Pigments, Biological; C042453:pyoverdin; D007501:Iron