Galline Ex-FABP Is an Antibacterial Siderocalin and a Lysophosphatidic Acid Sensor Functioning through Dual Ligand Specificities

Abstract

SUMMARY 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 1.8Å 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 co-purified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities.

Ligand binding‐dependent functions of the lipocalin NLaz: anin vivostudy inDrosophila

“…Also, Correnti et al . (39) have found that siderocalin binding pocket mutants that cannot bind siderophores in vitro lose their bacteriostatic activity when added to bacterial cultures.…”

Cation-π Interactions in Lipocalins: Structural and Functional Implications

“…Our calculations indicate that the cation-π interactions in pairs K125-Y132, F123-Lys134 and R81-W79, are energetically substantial enough (Table S1) to stabilize the holo-conformation of the protein. This is in addition to the cation-π bonds that were posited to occur with the ligand (9, 10). An alternative holo-conformation observed with a different ligand, carboxymycobactin T, (1×8U) is stabilized by the complementary cation-π in the pair R81-Y101 (Fig.…”

“…In lipocalins the study of cation-π interactions has been limited to two siderocalins, the neutrophil gelatinase-associated lipocalin (NGAL) and extracellular fatty acid binding protein (Ex-FABP). The analysis was restricted to the cation-π interaction in ligand recognition, i.e., only between ligand and protein side chains (9, 10). Here we will demonstrate that cation-π interactions within the protein can modulate ligand binding in a different way by stabilizing holo-form conformations in lipocalins.…”

Siderocalins: Siderophore binding proteins evolved for primary pathogen host defense

“…Ex-FABP is a 21 kDa lipocalin found in chickens [4,46]. The structural similarity to Scn, including a wide, positively charged calyx, prompted investigating the siderophore binding properties of Ex-FABP and the discovery that it is a siderocalin [47] (Figure 1).…”