Biochemical and Functional Evaluation of the Intramolecular Disulfide Bonds in the Zinc-Chelating Antimicrobial Protein Human S100A7 (Psoriasin)

Abstract

Human S100A7 (psoriasin) is a metal-chelating protein expressed by epithelial cells. It is a 22-kDa homodimer with two EF-hand domains per subunit, and two transition-metal-binding His3Asp sites at the dimer interface. Each subunit contains two cysteine residues that can exist as free thiols (S100A7red) or as an intramolecular disulfide bond (S100A7ox). Herein, we examine the disulfide bond redox behavior, the Zn(II) binding properties, and the antibacterial activity of S100A7, as well as the effect of Ca(II) ions on these properties. In agreement with prior work, (Hein et al. Proc. Natl. Acad. Sci. U. S. A. 2013, 112, 13039–13044), we show that apo S100A7ox is a substrate for the mammalian thioredoxin system; however, negligible reduction of the disulfide bond is observed for Ca(II)- and Zn(II)-bound S100A7ox. Furthermore, metal binding depresses the midpoint potential of the disulfide bond. S100A7ox and S100A7red each coordinate two equivalents of Zn(II) with sub-nanomolar affinity in the absence and presence of Ca(II) ions, and the cysteine thiolates in S100A7red do not form a third high-affinity Zn(II) site. These results refute a prior model implicating the Cys thiolates of S100A7red in high-affinity Zn(II) binding (Hein et al. Proc. Natl. Acad. Sci. U. S. A. 2013, 112, 13039–13044). S100A7ox and the disulfide-null variants show comparable Zn(II)-depletion profiles; however, only S100A7ox exhibits antibacterial activity against several bacterial species. Metal substitution experiments suggest that the disulfide bonds in S100A7 may enhance metal sequestration by the His3Asp sites and thereby confer growth inhibitory properties to S100A7ox.

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

“…10–12,14–17,20 Nevertheless, investigations of CP-Ser 20 as well as the host-defense proteins S100A7 34 and S100A12 35 have demonstrated that His 3 Asp sites sequester Zn(II). Moreover, studies of metal depletion from microbial growth media indicate that His 3 Asp sites select for Zn(II) over other metals, including Ni(II).…”

“…Moreover, studies of metal depletion from microbial growth media indicate that His 3 Asp sites select for Zn(II) over other metals, including Ni(II). 14,34,35 We therefore sought to examine metal exchange at the His 3 Asp site using the pull-down assay described above. Efforts to purify a biotinylated His 4 variant resulted in poor yields because the protein routinely precipitated, and we were unable to readily obtain sufficient quantities of this protein for the assay.…”

New Insights of the Zn(II)-Induced P2 × 4R Positive Allosteric Modulation: Role of Head Receptor Domain SS2/SS3, E160 and D170

“…Disulfide bond reduction may be assisted by extracellular glutathione, a tripeptide with antioxidant functions that is localized both intracellularly and extracellularly [44]. As a second option, we deem it to be possible that a fraction of the P2 × 4R head domain disulfide bonds may be in a redox balance between free SH and its oxidized form as demonstrated for psoriasin (S100A7), which is released from epithelial cells in two forms, one oxidized and another reduced [45]. We are aware that Zn(II) cannot reduce disulfides (as it does for instance metallic zinc [46]) but our data strongly supports that somehow the alkylation process depends on Zn(II).…”

S100 Proteins in Alzheimer’s Disease

“…S100 proteins occur mainly as homodimers (Barger et al, 1992; Giannakopoulos et al, 1996; Matsui Lee et al, 2000; Cunden et al, 2017). Specifically, it is known that calcium binding to S100 proteins triggers conformational changes that expose a hydrophobic cleft that is crucial to interaction with partners to their activation, regulation and signaling functions.…”