Divalent cations influence the dimerization mode of murine S100A9 protein by modulating its disulfide bond pattern

Abstract

The crystallographic structure of mS100A9 bound to calcium and zinc is reported A novel Zn-binding site and a disulfide bridge rigidify mS100A9 C-terminus In solution, mS100A9 exists both as non-covalent and disulfide-crosslinked homodimers Divalent cations modulate the relative proportion of the different mS100A9 homodimers 2 Abstract S100A9, with its congener S100A8, belongs to the S100 family of calcium-binding proteins found exclusively in vertebrates. These two proteins are major constituents of neutrophils. In response to a pathological condition, they can be released extracellularly and become alarmins that induce both pro-and anti-inflammatory signals, through specific cell surface receptors. They also act as antimicrobial agents, mainly as a S100A8/A9 heterocomplex, through metal sequestration. The mechanisms whereby divalent cations modulate the extracellular functions of S100A8 and S100A9 are still unclear. Importantly, it has been proposed that these ions may affect both the ternary and quaternary structure of these proteins, thereby influencing their physiological properties. In the present study, we report the crystal structures of WT and C80A murine S100A9 (mS100A9), determined at 1.45 and 2.35 Å resolution, respectively, in the presence of calcium and zinc. These structures reveal a canonical homodimeric form for the protein. They also unravel an intramolecular disulfide bridge that stabilizes the C-terminal tail in a rigid conformation, thus shaping a second Zn-binding site per S100A9 protomer. In solution, mS100A9 apparently binds only two zinc ions per homodimer, with an affinity in the micromolar range, and aggregates in the presence of excess zinc. Using mass spectrometry, we demonstrate that mS100A9 can form both non-covalent and covalent homodimers with distinct disulfide bond patterns. Interestingly, calcium and zinc seem to affect differentially the relative proportion of these forms. We discuss how the metal-dependent interconversion between mS100A9 homodimers may explain the versatility of physiological functions attributed to the protein.

Overexpression of S100A9 in obesity impairs macrophage differentiation via TLR4-NFkB-signaling worsening inflammation and wound healing

“…They detected S100A9 monomers, S100A8/S100A9 heterodimers and S100A9 homodimers intracellularly in spleen cells of tumor bearing animals while only S100A9 monomers and homodimers were secreted. Using mass spectrometry, non-covalent and covalent homodimers of mouse S100A9 have been identified 46 . Consistent to our data, Björk et al demonstrated that S100A9 binds to immobilized TLR4 while S100A8 shows only weak interaction 40 .…”

“…It becomes more and more evident that S100 proteins exist in vivo in multiple forms. This plasticity might contribute to their diverse and sometimes opposite functions 46 .…”

siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9

“…In addition to the Ca 2+ -binding EF-hand motifs involved in heterodimer formation, S100A8 and S100A9 are structurally characterized by a Zn 2+ -binding site that consists of the HxxxH motif ( Fig. S4 ) ( 22 , 23 ). Mouse S100A9 contains two HxxxH motifs close to the C terminus.…”

Molecular Basis of Ca(II)-Induced Tetramerization and Transition-Metal Sequestration in Human Calprotectin

“…Murine and rat S100A9 exhibits a cysteine (C111) in place of L109 (Figure C). A recent crystal structure of the murine S100A9 β 2 homodimer shows the formation of a disulfide bond between C91 and C111, suggesting that the tail is stabilized by disulfide bond formation. , Thus, it appears that tethering of the C-terminus via hydrophobic interactions or disulfide bonding is a common theme found in S100A9 orthologues and explains the tendency of L109 in CP to bind to this hydrophobic patch.…”

“…The tail appears intrinsically unstructured except for L109 that interacts with F48 and L86 of the same subunit. (C) In the crystal structure of the Zn(II)-and Ca(II)-bound murine β 2 homodimer (PDB code 6ZDY),67,68 the C-terminal tail ranges from residues N99 to K111. The disulfide bond formed by residues C91 and C109 is shown.…”