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

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 <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.