The Siderocalin/Enterobactin Interaction: A Link between Mammalian Immunity and Bacterial Iron Transport¹

Abstract

The siderophore enterobactin (Ent) is produced by enteric bacteria to mediate iron uptake. Ent scavenges iron and is taken up by the bacteria as the highly stable ferric complex [FeIII(Ent)]3−. This complex is also a specific target of the mammalian innate immune system protein, Siderocalin (Scn), which acts as an anti-bacterial agent by specifically sequestering siderophores and their ferric complexes during infection. Recent literature suggesting that Scn may also be involved in cellular iron transport has increased the importance of understanding the mechanism of siderophore interception and clearance by Scn; Scn is observed to release iron in acidic endosomes and [FeIII(Ent)]3− is known to undergo a change from catecholate to salicylate coordination in acidic conditions, which is predicted to be sterically incompatible with the Scn binding pocket (also referred to as the calyx). To investigate the interactions between the ferric Ent complex and Scn at different pH values, two recombinant forms of Scn with mutations in three residues lining the calyx were prepared: Scn-W79A/R81A and Scn-Y106F. Binding studies and crystal structures of the Scn-W79A/R81A:[FeIII(Ent)]3− and Scn-Y106F:[FeIII(Ent)]3− complexes confirm that such mutations do not affect the overall conformation of the protein but do weaken significantly its affinity for [FeIII(Ent)]3−. Fluorescence, UV-Vis and EXAFS spectroscopies were used to determine Scn/siderophore dissociation constants and to characterize the coordination mode of iron over a wide pH range, in the presence of both mutant proteins and synthetic salicylate analogs of Ent. While Scn binding hinders salicylate coordination transformation, strong acidification results in the release of iron and degraded siderophore. Iron release may therefore result from a combination of Ent degradation and coordination change.

α–Intercalated cells defend the urinary system from bacterial infection

“…In fact, the LCN2:Ent:Fe 3+ complex is stable at pH 4.0, which implies that LCN2 can sequester Ent:Fe 3+ in most biological fluids, even acidified urine (8). Fe can only be released from the LCN2:Ent:Fe 3+ complex in acidified lysosomes, following the reduction of Fe and the proteolytic cleavage of LCN2 (42). Hence, LCN2 arrests extracellular bacterial growth by chelating Ent:Fe 3+ , limiting its bioavailability in solution (40,43,44).…”

Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides

“…The general recognition mechanism of Scn has been extensively studied and is well understood (22,24,(27)(28)(29)(30)(31)(32)(33). Ligands bind in the highly sculpted protein calyx, where the key interaction is through an aryl group binding in a snug pocket between the side chains of K125 and K134, with electrostatic and cation-π interactions to this ring and neighboring ones mediated by the side chains of R81, K124, and K134 (Fig.…”

“…2 A-C) show clear electron density for the metal, but the only part of the hexadentate chelator that was resolvable is the catechol moiety binding in the K125/K134 pocket. Prior structural analyses had shown that [Fe III (Ent)] 3-is susceptible to backbone hydrolysis in the Scn calyx (27), but TRENCAM is nonhydrolyzable, so partial disordering of Ent or TRENCAM in these structures is likely accounted for by a combination of the inability of these tightly constrained chelators to fully coordinate ions larger than Fe 3+ , and the inability of the tightly constrained calyx to accommodate equatorial expansion of the metal-chelator complex. This latter property is consistent with the inability of Scn to accommodate salicylate-mode binding ferric siderophores (27), and explains the observation of an uncoordinated catechol group in the [Th IV (TRENCAM)] 2-complex structure (Fig.…”

An RNA Aptamer-Based Microcantilever Sensor To Detect the Inflammatory Marker, Mouse Lipocalin-2

“…The three calyx alanine substitutions were shown via circular dichroism (CD) to have little overall effect on mLcn2 structure (see Figure S6 in the Supporting Information), which is a conclusion supported by a crystallography study of hLcn2 with double mutations that included Arg103 in hLcn2. 7 In addition, the calyx mutants were evaluated for structural integrity by gel filtration and native polyacrylamide gel electrophoresis (see Figure S7 and Table S2 in the Supporting Information). These results show that, although there are differences between the mutants and WT proteins in the gel filtration elution profiles and in their rates of travel on electrophoresis, the differences are small and do not correlate with the ability of the aptamer to bind the protein.…”