Human calprotectin affects the redox speciation of iron

Abstract

We report that the metal-sequestering human host-defense protein calprotectin (CP, S100A8/S100A9 oligomer) affects the redox speciation of iron (Fe) in bacterial growth media and buffered aqueous solution. Under aerobic conditions and in the absence of an exogenous reducing agent, CP-Ser (S100A8(C42S)/S100A9(C3S) oligomer) depletes Fe from three different bacterial growth media preparations over a 48-h timeframe (T = 30 °C). The presence of the reducing agent β-mercaptoethanol accelerates this process and allows CP-Ser to deplete Fe over a ≈1-h timeframe. Fe-depletion assays performed with metal-binding-site variants of CP-Ser show that the hexahistidine (His6) site, which coordinates Fe(II) with high affinity, is required for Fe depletion. An analysis of Fe redox speciation in buffer containing Fe(III) citrate performed under aerobic conditions demonstrates that CP-Ser causes a time-dependent increase in the [Fe(II)]/[Fe(III)] ratio. Taken together, these results indicate that the hexahistidine site of CP stabilizes Fe(II) and thereby shifts the redox equilibrium of Fe to the reduced ferrous state under aerobic conditions. We also report that the presence of bacterial metabolites affects the Fe-depleting activity of CP-Ser. Supplementation of bacterial growth media with an Fe(III)-scavenging siderophore (enterobactin, staphyloferrin B, or desferrioxamine B) attenuates the Fe-depleting activity of CP-Ser. This result indicates that formation of Fe(III)-siderophore complexes blocks CP-mediated reduction of Fe(III) and hence the ability of CP to coordinate Fe(II). In contrast, the presence of pyocyanin (PYO), a redox-cycling phenazine produced by Pseudomonas aeruginosa that reduces Fe(III) to Fe(II), accelerates Fe depletion by CP-Ser under aerobic conditions. These findings indicate that the presence of microbial metabolites that contribute to metal homeostasis at the host/pathogen interface can affect the metal-sequestering function of CP.

A unique ferrous iron binding mode is associated with large conformational changes for the transport protein FpvC of Pseudomonas aeruginosa

“…The protein calprotectin has been shown to be a polyspecific metal protein, able to bind Mn 2+ , Ni 2+ , Zn 2+ and Fe 2+ in the hexahistidine site [23,31]. Its structure with a Ni 2+ bound to the hexahistidine motif was reported as unique in 2017 among structurally characterized nickel proteins [23].…”

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His3Asp Site

“…1–5 CP exhibits broad-spectrum antimicrobial activity attributed to its ability to sequester essential metal nutrients from invading pathogens. 1,4,6,7 Human CP coordinates divalent first-row transition metals, including Mn(II), 8–13 Fe(II), 14–16 Ni(II), 17 Cu(II), 18 and Zn(II), 8,19,20 with sufficiently high affinity to prevent microbial acquisition of these nutrients.…”

Preparation and Iron Redox Speciation Study of the Fe(II)-Binding Antimicrobial Protein Calprotectin

“…For the Fe speciation assay, ferrozine is used to determine the Fe(II) content of solutions via detection of the [Fe(ferrozine)3] 4- complex by optical absorption spectroscopy (19, 27, 28). …”

“…Site 2 is a His 6 motif composed of (A8)His17, (A8)His27, (A9)His91, (A9)His95, (A9)His103 and (A9)His105(13–15). The His 6 site of hCP has gained significant attention in recent years because it can sequester a range of divalent first-row transition metal ions including Mn(II), Fe(II), Zn(II) and Ni(II)(1, 14–17, 19, 20). Like the human protein, murine CP (mCP) is also a heterooligomer of S100A8 and S100A9, and each subunit contains a canonical C-terminal EF-hand and a non-canonical N-terminal EF-hand domain.…”

“…These protocols are based on published work (12, 21), provide each protein as the apo heterodimer, and can be used for preparing native hCP as well as other mCP and hCP-Ser variants with single or multiple point mutations. Because the His 6 site of hCP was recently shown to sequester Fe(II) and the protein was found to affect the redox speciation of Fe in aerobic solution (18, 19), we also provide a protocol for an Fe speciation assay that we first designed and utilized to study hCP-Ser(19). Herein, we extend this assay to mCP and report that this protein also shifts the redox speciation of Fe from Fe(III) to Fe(II) under aerobic conditions.…”