2 research outputs found
Identification of a Positively Charged Platform in <i>Staphylococcus aureus</i> HtsA That Is Essential for Ferric Staphyloferrin A Transport
In response to iron starvation, <i>Staphylococcus aureus</i> secretes both staphyloferrin A and
staphyloferrin B, which are high-affinity
iron-chelating molecules. The structures of both HtsA and SirA, the
ferric-staphyloferrin A [FeĀ(III)-SA] and ferric-staphyloferrin B [FeĀ(III)-SB]
receptors, respectively, have recently been determined. The structure
of HtsA identifies a novel form of ligand entrapment composed of many
positively charged residues. Through ionic interactions, the binding
pocket appears highly adapted for the binding of the highly anionic
siderophore SA. However, biological validation of the importance of
the nine SA-interacting residues (six arginines, one tyrosine, one
histidine, and one lysine) has not been previously performed. Here,
we mutated each of the FeĀ(III)-SA-interacting residues in HtsA and
found that substitutions R104A, R126A, H209A, R306A, and R306K resulted
in a reduction of binding affinity of HtsA for FeĀ(III)-SA. While mutation
of almost all proposed ligand-interacting residues decreased the ability
of <i>S. aureus</i> cells to transport <sup>55</sup>FeĀ(III)-SA, <i>S. aureus</i> expressing HtsA R104A, R126A, R306A, and R306K
showed the greatest transport defects and were incapable of growth
in iron-restricted growth media in a SA-dependent manner. These three
residues cluster together and, relative to other residues in the binding
pocket, move very little between the apo and closed holo structures.
Their essentiality for receptor function, together with structural
information, suggests that they form a positively charged platform
that is required for initial contact with the terminal carboxyl groups
of the two citrates in the FeĀ(III)-SA complex. This is a likely mechanism
by which HtsA discerns iron-bound SA from iron-free SA
An ABC Transporter with Two Periplasmic Binding Proteins Involved in Iron Acquisition in <i>Pseudomonas aeruginosa</i>
Pyoverdine I is the main siderophore secreted by <i>Pseudomonas aeruginosa</i> PAO1 to obtain access to iron. After
extracellular iron chelation, pyoverdine-Fe uptake into the bacteria
involves a specific outer-membrane transporter, FpvA. Iron is then
released in the periplasm by a mechanism involving no siderophore
modification but probably iron reduction. The proteins involved in
this dissociation step are currently unknown. The pyoverdine locus
contains the <i>fpvCDEF</i> operon, which contains four
genes. These genes encode an ABC transporter of unknown function with
the distinguishing characteristic of encompassing two periplasmic
binding proteins, FpvC and FpvF, associated with the ATPase, FpvE,
and the permease, FpvD. Deletion of these four genes partially inhibited
cytoplasmic uptake of <sup>55</sup>Fe in the presence of pyoverdine
and markedly slowed down the <i>in vivo</i> kinetics of
iron release from the siderophore. This transporter is therefore involved
in iron acquisition by pyoverdine in <i>P. aeruginosa</i>. Sequence alignments clearly showed that FpvC and FpvF belong to
two different subgroups of periplasmic binding proteins. FpvC appears
to be a metal-binding protein, whereas FpvF has homology with ferrisiderophore
binding proteins. <i>In vivo</i> cross-linking assays and
incubation of purified FpvC and FpvF proteins showed formation of
complexes between both proteins. These complexes were able to bind <i>in vitro</i> PVDI-Fe, PVDI-Ga, or apo PVDI. This is the first
example of an ABC transporter involved in iron acquisition <i>via</i> siderophores, with two periplasmic binding proteins
interacting with the ferrisiderophore. The possible roles of FpvCDEF
in iron uptake by the PVDI pathway are discussed