Abstract
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 Escherichia coli siderophore. Pathogenic E. coli 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.