Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.

The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal (also known as neutrophil gelatinase associated lipocalin, siderocalin, lipocalin 2) sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH-sensitive mechanism. As catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal-catechol-Fe(III) complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal-siderophore interactions but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.

Enzymatic hydrolysis of trilactone siderophores: where chiral recognition occurs in enterobactin and bacillibactin iron transport.

Bacillibactin and enterobactin are hexadentate catecholate siderophores produced by bacteria upon iron limitation to scavenge ferric ion and seem to be the ultimate siderophores of their two respective domains: Gram-positive and Gram-negative. Iron acquisition mediated by these trilactone-based ligands necessitates enzymatic hydrolysis of the scaffold for successful intracellular iron delivery. The esterases BesA and Fes hydrolyze bacillibactin and enterobactin, respectively, as well as the corresponding iron complexes. Bacillibactin binds iron through three 2,3-catecholamide moieties linked to a trithreonine scaffold via glycine spacers, whereas in enterobactin the iron-binding moieties are directly attached to a tri-l-serine backbone; although apparently minor, these structural differences result in markedly different iron coordination properties and iron transport behavior. Comparison of the solution thermodynamic and circular dichroism properties of bacillibactin, enterobactin and the synthetic analogs d-enterobactin, SERGlyCAM and d-SERGlyCAM has determined the role of each different feature in the siderophores' molecular structures in ferric complex stability and metal chirality. While opposite metal chiralities in the different complexes did not affect transport and incorporation in Bacillus subtilis, ferric complexes formed with the various siderophores did not systematically promote growth of the bacteria. The bacillibactin esterase BesA is less specific than the enterobactin esterase Fes; BesA can hydrolyze the trilactones of both siderophores, while only the tri-l-serine trilactone is a substrate of Fes. Both enzymes are stereospecific and cannot cleave tri-d-serine lactones. These data provide a complete picture of the microbial iron transport mediated by these two siderophores, from initial recognition and transport to intracellular iron release.

Parsing the functional specificity of Siderocalin/Lipocalin 2/NGAL for siderophores and related small-molecule ligands.

Siderocalin/Lipocalin 2/Neutrophil Gelatinase Associated Lipocalin/24p3 is an innate immune system protein with bacteriostatic activity, acting by tightly binding and sequestering diverse catecholate and mixed-type ferric siderophores from enteric bacteria and mycobacteria. Bacterial virulence achieved through siderophore modifications, or utilization of alternate siderophores, can be explained by evasion of Siderocalin binding. Siderocalin has also been implicated in a wide variety of disease processes, though often in seemingly contradictory ways, and has been proposed to bind to a broader array of ligands beyond siderophores. Using structural, directed mutational, and binding studies, we have sought to rigorously test, and fully elucidate, the Siderocalin recognition mechanism. Several proposed ligands fail to meet rigorous binding criteria, including the bacterial siderophore pyochelin, the iron-chelating catecholamine hormone norepinephrine, and the bacterial second messenger cyclic diguanylate monophosphate. While possessing a remarkably rigid structure, in principle simplifying analyses of ligand recognition, understanding Scn recognition is complicated by the observed conformational and stoichiometric plasticity, and instability, of its bona fide siderophore ligands. Since the role of Siderocalin at the early host/pathogen interface is to compete for bacterial ferric siderophores, we also analyzed how bacterial siderophore binding proteins and enzymes alternately recognize siderophores that efficiently bind to, or evade, Siderocalin sequestration - including determining the crystal structure of Bacillus cereus YfiY bound to schizokinen. These studies combine to refine the potential physiological functions of Siderocalin by defining its multiplexed recognition mechanism.

The role of electrostatics in siderophore recognition by the immunoprotein Siderocalin.

Iron is required for virulence of most bacterial pathogens, many of which rely on siderophores, small-molecule 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 analogues, the contribution of electrostatic interactions, including both charge-charge and cation-pi, to the recognition of 2,3-catecholate siderophores has been deconvoluted. The analogues 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 (tren) 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 analogue series was determined through small-molecule crystallography, and the binding interactions were 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 analogues 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.

Immune interference in Mycobacterium tuberculosis intracellular iron acquisition through siderocalin recognition of carboxymycobactins.

The innate immune system antibacterial protein Siderocalin (Scn) binds ferric carboxymycobactin (CMB) and also several catecholate siderophores. Although the recognition of catecholates by Scn has been thoroughly investigated, the binding interactions of Scn with the full spectrum of CMB isoforms have not been studied. Here we show that Scn uses different binding modes for the limited subset of bound CMB isoforms, resulting in a range of binding affinities that are much weaker than other siderophore targets of Scn. Understanding the binding interaction between Scn and CMBs provides clues for the influence of Scn on mycobacterial iron acquisition.

Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities.

Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in vitro under iron-limiting conditions. The 1.8 Å crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding copurified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities.

Anthrax pathogen evades the mammalian immune system through stealth siderophore production.

Systemic anthrax, caused by inhalation or ingestion of Bacillus anthracis spores, is characterized by rapid microbial growth stages that require iron. Tightly bound and highly regulated in a mammalian host, iron is scarce during an infection. To scavenge iron from its environment, B. anthracis synthesizes by independent pathways two small molecules, the siderophores bacillibactin (BB) and petrobactin (PB). Despite the great efficiency of BB at chelating iron, PB may be the only siderophore necessary to ensure full virulence of the pathogen. In the present work, we show that BB is specifically bound by siderocalin, a recently discovered innate immune protein that is part of an antibacterial iron-depletion defense. In contrast, neither PB nor its ferric complex is bound by siderocalin. Although BB incorporates the common 2,3-dihydroxybenzoyl iron-chelating subunit, PB is novel in that it incorporates the very unusual 3,4-dihydroxybenzoyl chelating subunit. This structural variation results in a large change in the shape of both the iron complex and the free siderophore that precludes siderocalin binding, a stealthy evasion of the immune system. Our results indicate that the blockade of bacterial siderophore-mediated iron acquisition by siderocalin is not restricted to enteric pathogenic organisms and may be a general defense mechanism against several different bacterial species. Significantly, to evade this innate immune response, B. anthracis produces PB, which plays a key role in virulence of the organism. This analysis argues for antianthrax strategies targeting siderophore synthesis and uptake.