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

ABSTRACT: 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.

PROVIDER: S-EPMC2907470 | BioStudies |

REPOSITORIES: biostudies

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Site-Specific Siderocalin Binding to Ferric and Ferric-Free Enterobactin As Revealed by Mass Spectrometry.

Project description:Both host and pathogen competitively manipulate coordination environments during bacterial infections. Human cells release the innate immune protein siderocalin (Scn, also known as lipocalin-2/Lcn2, neutrophil gelatinase-associated lipocalin/NGAL) that can inhibit bacterial growth by sequestering iron in a ferric complex with enterobactin (Ent), the ubiquitous <i>Escherichia coli</i> siderophore. Pathogenic <i>E. coli</i> use the virulence-associated esterase IroE to linearize the Ent cyclic trilactone to linear enterobactin (lin-Ent). We characterized lin-Ent interactions with Scn by using native mass spectrometry (MS) with hydrogen-deuterium exchange (HDX) and Lys/Arg specific covalent footprinting. These approaches support 1:1 binding of both Fe(III)-lin-Ent to Scn and iron-free lin-Ent to Scn. Both ferric and nonferric lin-Ent localize to all three pockets of the Scn calyx, consistent with Scn capture of lin-Ent both before and after Fe(III) chelation. These findings raise the possibility that Scn neutralizes both siderophores and siderophore-bound iron during infections. This integrated, MS-based approach circumvents the limitations that frustrate traditional structural approaches to examining Scn interactions with enterobactin-based ligands.

| S-EPMC7236765 | BioStudies

Purification and Structural Characterization of "Simple Catechol", the NGAL-Siderocalin Siderophore in Human Urine.

Project description:The identification of ligands that bind the protein Neutrophil Gelatinase-Associated Lipocalin (NGAL, Siderocalin, Lipocalin-2) have helped to elucidate its function. NGAL-Siderocalin binds and sequesters the iron loaded bacterial siderophore enterochelin (Ent), defining the protein as an innate immune effector. Simple metabolic catechols can also form tight complexes with NGAL-Siderocalin and ferric iron, suggesting that the protein may act as an iron scavenger even in the absence of Ent. While different catechols have been detected in human urine, they have not been directly purified from a biofluid and demonstrated to ligate iron with NGAL-Siderocalin. This paper describes a "natural products" approach to identify small molecules that mediate iron binding to NGAL-Siderocalin. A 10K filtrate of human urine was subjected to multiple steps of column chromatography and reverse-phase HPLC, guided by NGAL-Siderocalin-iron binding assays and LC-MS detection. The co-factor forming a ternary structure with iron and NGAL-Siderocalin was identified as authentic simple catechol (dihydroxybenze) by ESI-HR-Mass, UV, and NMR spectrometric analysis. Comparison of the binding strengths of different catechols demonstrated that the vicinal-dihydroxyl groups were the key functional groups and that steric compatibilities of the catechol ring have the strongest effect on binding. Although catechol was a known NGAL-Siderocalin co-factor, our purification directly confirmed its presence in urine as well as its capacity to serve as an iron trap with NGAL-Siderocalin.

| S-EPMC4527557 | BioStudies

Siderocalin outwits the coordination chemistry of vibriobactin, a siderophore of Vibrio cholerae.

Project description:The human protein siderocalin (Scn) inhibits bacterial iron acquisition by binding catechol siderophores. Several pathogenic bacteria respond by making stealth siderophores that are not recognized by Scn. Fluvibactin and vibriobactin, respectively of Vibrio fluvialis and Vibrio cholerae , include an oxazoline adjacent to a catechol. This chelating unit binds iron either in a catecholate or a phenolate-oxazoline coordination mode. The latter has been suggested to make vibriobactin a stealth siderophore without directly identifying the coordination mode in relation to Scn binding. We use Scn binding assays with the two siderophores and two oxazoline-substituted analogs and the crystal structure of Fe-fluvibactin:Scn to show that the oxazoline does not prevent Scn binding; hence, vibriobactin is not a stealth siderophore. We show that the phenolate-oxazoline coordination mode is present at physiological pH and is not bound by Scn. However, Scn binding shifts the coordination to the catecholate mode and thereby inactivates this siderophore.

| S-EPMC3783644 | BioStudies

The v-myc-induced Q83 lipocalin is a siderocalin.

Project description:Siderocalins are atypical lipocalins able to capture siderophores with high affinity. They contribute to the innate immune response by interfering with bacterial siderophore-mediated iron uptake but are also involved in numerous physiological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. The Q83 lipocalin was originally identified based on its overexpression in quail embryo fibroblasts transformed by the v-myc oncogene. We show here that Q83 is a siderocalin, binding the siderophore enterobactin with an affinity and mode of binding nearly identical to that of neutrophil gelatinase-associated lipocalin (NGAL), the prototypical siderocalin. This strengthens the role of siderocalins in cancer progression and inflammation. In addition, we also present the solution structure of Q83 in complex with intact enterobactin and a detailed analysis of the Q83 binding mode, including mutagenesis of the critical residues involved in enterobactin binding. These data provide a first insight into the molecular details of siderophore binding and delineate the common molecular properties defining the siderocalin protein family.

| S-EPMC3009892 | BioStudies

Human Metabolome-derived Cofactors Are Required for the Antibacterial Activity of Siderocalin in Urine.

Project description:In human urinary tract infections, host cells release the antimicrobial protein siderocalin (SCN; also known as lipocalin-2, neutrophil gelatinase-associated lipocalin, or 24p3) into the urinary tract. By binding to ferric catechol complexes, SCN can sequester iron, a growth-limiting nutrient for most bacterial pathogens. Recent evidence links the antibacterial activity of SCN in human urine to iron sequestration and metabolomic variation between individuals. To determine whether these metabolomic associations correspond to functional Fe(III)-binding SCN ligands, we devised a biophysical protein binding screen to identify SCN ligands through direct analysis of human urine. This screen revealed a series of physiologic unconjugated urinary catechols that were able to function as SCN ligands of which pyrogallol in particular was positively associated with high urinary SCN activity. In a purified, defined culture system, these physiologic SCN ligands were sufficient to activate SCN antibacterial activity against Escherichia coli In the presence of multiple SCN ligands, native mass spectrometry demonstrated that SCN may preferentially combine different ligands to coordinate iron, suggesting that availability of specific ligand combinations affects in vivo SCN antibacterial activity. These results support a mechanistic link between the human urinary metabolome and innate immune function.

| S-EPMC5207064 | BioStudies

Siderocalin/Lcn2/NGAL/24p3 does not drive apoptosis through gentisic acid mediated iron withdrawal in hematopoietic cell lines.

Project description:Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.

| S-EPMC3424236 | BioStudies

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

Project description: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.

| S-EPMC7337064 | BioStudies

The Ligands of Neutrophil Gelatinase-Associated Lipocalin.

Project description:Neutrophil gelatinase associated lipocalin (NGAL), was originally identified in neutrophil granules as a heterodimer complex with gelatinase B (matrix metalloproteinase 9, MMP9), but more recently has been found to be secreted by damaged epithelial cells. Ngal is a member of the lipocalin family and subsequently named as lipocalin 2 on the basis of structural similarity with other members of the lipocalin family and its potential association with hydrophobic retinol and cholesterol oleate more strongly than their hydrophilic counterparts. In 2002, a landmark paper suggested that Ngal is a bacteriostatic agent which blocks iron acquisition by interacting with a number of bacterial siderophores, especially enterobactin. Since then, more siderophore-carrying functions have been reported than the possibility of hydrophobic ligand transport. In this setting, Ngal was renamed Siderocalin. Functions of siderocalin include not only bacteriostatic activity but potentially as a mediator of cell growth and differentiation; some of these functions appear to be referable to the holo siderocalin:siderophore:iron complex and recent work suggests that metabolic products may act as mammalian siderophores bound by Ngal. While still speculative, it may be that the mammalian siderophores can establish the missing link between Ngal and a number of its functions in vivo. This review provides an overview of the discoveries of the different ligands of Ngal and consequently related functions. Hydrophobic ligands, bacterial siderophores as well as their modified structures (synthetic siderophores), and mammalian siderophores are summarized.

| S-EPMC5014392 | BioStudies

The siderocalin/enterobactin interaction: a link between mammalian immunity and bacterial iron transport.

Project description: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 [Fe (III)(Ent)] (3-). This complex is also a specific target of the mammalian innate immune system protein, Siderocalin (Scn), which acts as an antibacterial 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 [Fe (III)(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:[Fe (III)(Ent)] (3-) and Scn-Y106F:[Fe (III)(Ent)] (3-) complexes confirm that such mutations do not affect the overall conformation of the protein but do weaken significantly its affinity for [Fe (III)(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 analogues 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.

| S-EPMC3188318 | BioStudies

Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17.

Project description:The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL's siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (?10 ?m) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.

| S-EPMC4742754 | BioStudies

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