The Role of Electrostatics in Siderophore Recognition by the Immunoprotein Siderocalin¹

Abstract

Iron is required for virulence of most bacterial pathogens, many of which rely on siderophores, smallmolecule chelators, to scavenge iron in mammalian hosts. As an immune response, the human protein Siderocalin binds both apo and ferric siderophores in order to intercept delivery of iron to the bacterium, impeding virulence. The introduction of steric clashes into the siderophore structure is an important mechanism of evading sequestration. However, in the absence of steric incompatibilities, electrostatic interactions determine siderophore strength of binding by Siderocalin. By using a series of isosteric enterobactin analogs, the contribution of electrostatic interactions, including both chargecharge and cation-π, to the recognition of 2,3-catecholate siderophores has been deconvoluted. The analogs used in the study incorporate a systematic combination of 2,3-catecholamide (CAM) and N-hydroxypyridinonate (1,2-HOPO) binding units on a tris(2-aminoethyl)amine backbone, [tren (CAM) m (1,2-HOPO) n where m = 0, 1, 2, or 3 and n = 3 -m]. The shape complementarity of the synthetic analog series was determined through small molecule crystallography and the binding interactions investigated through a fluorescence-based binding assay. These results were modeled and correlated through ab initio calculations of the electrostatic properties of the binding units. Although all the analogs are accommodated in the binding pocket of Siderocalin, the ferric complexes incorporating decreasing numbers of CAM units are bound with decreasing affinities (K d = >600, 43, 0.8 and 0.3 nM for m = 0-3). These results elucidate the role of electrostatics in the mechanism of siderophore recognition by Siderocalin.

The v-myc-induced Q83 Lipocalin Is a Siderocalin

“…However, the K D of the Q83/Enterobactin complex is clearly within the range of 1 nM. Furthermore, a very similar enterobactin dissociation constant (K D of 0.41 nM) was reported for NGAL (25). 3Ϫ complex was solved.…”

“…It is possible that the effect of the mutation is larger than observed, since the affinity of the wild-type protein for enterobactin is less well determined than that of the weak-binding mutants. Nevertheless, a similar weakening of the affinity was observed for NGAL (25) when titrated by synthetic enterobactin analogues especially designed to disrupt one cation-interaction. Consequently, Q83 and NGAL seem to have very similar binding mode and recognition mechanism toward enterobactin.…”

“…Ent (Fig. 1A) is a tri-catechol derivative of a cyclic tri-serine lactone (21,22 (24,25). Ferric enterobactin rapidly degrades into dihydroxybenzoyl-serine (DHBS) and dihydroxybenzoic acid (DHBA).…”

Neutrophil Gelatinase-Associated Lipocalin Expresses Antimicrobial Activity by Interfering withl-Norepinephrine-Mediated Bacterial Iron Acquisition

“…However, as we were able to detect the binding of Fe III ⅐ (NE) 3 to NGAL in solution and also observed the coloring of protein crystals upon soaking, it is possible that the precise arrangement of the substituents protruding from the aromatic ring plays a more important role than previously assumed (27). In fact, the modeling of Fe III ⅐ (NE) 3 indicates that at least the lower part of this complex, which should reside at the bottom of the ligand pocket considering the crystal structure with bound Fe III ⅐ Ent (25), has a rather similar molecular shape and probably can undergo the same interactions with NGAL (Fig.…”

The Staphylococcus aureus Siderophore Receptor HtsA Undergoes Localized Conformational Changes to Enclose Staphyloferrin A in an Arginine-rich Binding Pocket

“…The crystal structures show that two Lys residues and a single Arg mediate binding to the negatively charged catecholates. Recently, it was established that electrostatic interactions between ferric siderophores and siderocalin are the prime determinants of binding affinity (81).…”