The Structure−Activity Relationship of Ferric Pyoverdine Bound to Its Outer Membrane Transporter:  Implications for the Mechanism of Iron Uptake

Abstract

Under iron limitation, Pseudomonas aeruginosa ATCC 15692 secretes a major siderophore, pyoverdine I (PvdI). This molecule chelates iron in the extracellular medium and shuttles it into the cells via a specific outer membrane transporter, FpvAI. PvdI consists of a fluorescent chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and containing one of the bidentate groups involved in iron chelation, linked to a peptide moiety containing the two other bidentate groups required for binding to Fe(3+). Kinetic studies, based on the fluorescence properties of this siderophore, showed that pH 8.0 was optimal for the binding of PvdI and PvdI-Fe to FpvAI. We investigated the mechanism of interaction of PvdI and PvdI-Fe with FpvAI, by synthesizing various analogues of this siderophore, determining their affinity for FpvAI in vitro and in vivo and their ability to transport iron, and interpreting the results obtained in light of the structure of FpvAI-PvdI. Our findings demonstrate that the succinyl moiety linked to the chromophore of PvdI and the first amino acid of the peptide moiety can be sterically hindered with no effect on binding or the iron uptake properties of PvdI-Fe. Moreover, the sequence and the structure of the peptide moiety of PvdI seems to be more important for the iron uptake step than for the binding of the siderophore to FpvAI. Finally, the efficiency of iron uptake and of recycling of the various PvdI analogues after iron release suggests that iron dissociates from PvdI on FpvAI or in the periplasm. All these data have serious implications for the specificity and mechanism of PvdI-mediated iron transport in P. aeruginosa.

Structure–function relationships in the bifunctional ferrisiderophore FpvA receptor from Pseudomonas aeruginosa

“…It is only able to bind the Pvd shown in Fig. 1a and a similar Pvd produced by a strain of P. fluorescens (Schons et al 2005). In FpvA-Pvd-Fe, Pvd-Fe is attached to the plug domain via the chromophore (Fig.…”

“…The peptide moiety, which determines the receptor specificity of the Pvd, interacts mainly with the b-barrel domain and the extracellular loops, rather than with the plug domain (Wirth et al 2007). Binding studies with different Pvd analogues have shown that Ser 1 and the succinyl moiety linked to the chromophore of Pvd can be sterically hindered with no effect on binding or the iron uptake properties of Pvd-Fe (Schons et al 2005). However, the second position of the peptide moiety in Pvd (Arg) cannot be subject to steric hindrance without a major decrease in affinity of the ferric-siderophore (Schons et al 2005).…”

“…Binding studies with different Pvd analogues have shown that Ser 1 and the succinyl moiety linked to the chromophore of Pvd can be sterically hindered with no effect on binding or the iron uptake properties of Pvd-Fe (Schons et al 2005). However, the second position of the peptide moiety in Pvd (Arg) cannot be subject to steric hindrance without a major decrease in affinity of the ferric-siderophore (Schons et al 2005). In FpvA-Fe-Pvd, the Arg of Pvd points toward the plug domain and is covered by part of loop L7 and interacts with D597 of this loop.…”

The Metal Dependence of Pyoverdine Interactions with Its Outer Membrane Receptor FpvA

“…We introduced a primary amine to Pvd via a PEG linker on the succinate moiety in order to be able to immobilize Pvd to a Biacore sensor chip by primary amine coupling. The succinate moiety was chosen as the attachment site because it has been previously demonstrated that the attachment of bulky groups to this part of Pvd had no effect on iron chelation and transport (27). Using the immobilized Pvd surface, we were able to compare the affinities of FpvA for Pvd and Pvd-Fe in a controlled and quantitative manner.…”

Unexpected Interaction of a Siderophore with Aluminum and Its Receptor

“…Apo-pyoverdine was described to be bound to the surface of the FpvA receptor in a pocket lined with aromatic residues (3,13,17). It has therefore been proposed that iron-loaded pyoverdine can exchange with unloaded pyoverdine at the surface of the receptor, a process which is dependent on the inner membrane protein TonB, which relays the proton motive force to FpvA (16). Here, Greenwald et al reveal that in reality apo-pyoverdine has no affinity at all for the FpvA receptor and that the previously observed fluorescence rather is due to the association of Al-pyoverdine with the receptor, meaning that the interactions measured by fluorescent resonance energy transfer are in fact those occurring between Al-pyoverdine and FpvA (12,13).…”