Calcium-dependent Tetramer Formation of S100A8 and S100A9 is Essential for Biological Activity

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S100 proteins comprise the largest family of calcium-binding proteins. Members of this family usually form homo- or heterodimers, which may associate to higher-order oligomers in a calcium-dependent manner. The heterodimers of S100A8 and S100A9 represent the major calcium-binding proteins in phagocytes. Both proteins regulate migration of these cells via modulation of tubulin polymerization. Calcium binding induces formation of (S100A8/S100A9)2 tetramers. The functional relevance of these higher-order oligomers of S100 proteins, however, is not yet clear. To investigate the importance of higher-order oligomerization for S100 proteins, we created a set of mutations within S100A9 (N69A, E78A, N69A+E78A) destroying the high-affinity C-terminal calcium-binding site (EF-hand II). Mutations in EF-hand II did not interfere with formation of the S100A8/S100A9 heterodimer as demonstrated by yeast two-hybrid experiments and pull-down assays. In contrast, mass spectrometric analysis and density gradient centrifugation revealed that calcium-induced association of (S100A8/S100A9)2 tetramers was strictly dependent on a functional EF-hand II in S100A9. Failure of tetramer formation was associated with a lack of functional activity of S100A8/S100A9 complexes in promoting the formation of microtubules. Thus, our data demonstrate that calcium-dependent formation of (S100A8/S100A9)2 tetramers is an essential prerequisite for biological function. This is the first report showing a functional relevance of calcium-induced higher-order oligomerization in the S100 family.

Introduction

S100A8 (myeloid related protein 8, MRP8) and S100A9 (MRP14) belong to the S100 family of calcium-binding proteins. Both proteins are specifically expressed in circulating neutrophils and early differentiation stages of monocytes,1 as well as in keratinocytes and epithelial cells under inflammatory conditions.2, 3 Like other S100 proteins, S100A8 and S100A9 are characterized by two calcium-binding sites of the EF-hand type with different affinities to bind calcium; a high affinity site at the C terminus (C-terminal EF-hand; EF-hand II) and a low affinity site at the N terminus (N-terminal EF-hand; EF-hand I).4, 5 Under physiological conditions, most S100 proteins form non-covalently associated complexes as a prerequisite for their biological functions. With the exception of the monomeric calbindin D9k,6 the majority of S100 proteins form homodimers, while heterodimerization is only described for some family members.7, 8, 9, 10, 11, 12 Moreover, there is increasing evidence that S100 proteins are able to form higher-order oligomers.13, 14, 15, 16 However, the functional relevance of these higher S100 complexes is not yet clear.

While it is commonly accepted that S100A8 and S100A9 form stable heterodimers in the absence of calcium, the oligomerization state of both proteins in the presence of calcium has long been contradictorily discussed in the literature; monomers, heterodimers, homodimers and trimers have been described earlier. Finally, structural analysis, yeast two-hybrid experiments and mass spectrometric studies clearly show that the heterodimer is the basic form of S100A8 and S100A9, which tetramerizes upon calcium-binding, confirming that the oligomerization state of both proteins in the presence of calcium is a (S100A8/S100A9)2 heterotetramer that binds eight Ca2+.16, 17, 18, 19

Calcium-induced complexes of S100A8 and S100A9 have been shown to colocalize with microtubules (MTs) during activation of monocytes.20 Functional analyses demonstrated that the complexes are involved in cytoskeletal organization and that they directly bind to tubulin and promote tubulin polymerization in a calcium-dependent manner.21 However, it is unclear whether the calcium-dependent induction of (S100A8/S100A9)2 tetramers plays a functional role in this modulating effect on MTs.

Since the formation of non-covalently associated (S100A8/S100A9)2 heterotetramer complexes is known to be calcium-dependent, we analyzed the importance of calcium-binding to the high affinity C-terminal EF-hand by creating specific S100A9 mutants. In analogy to the mutations in the C-terminal EF-hand structure of S100A10, which is unable to bind calcium,22 we replaced the homologous amino acid residues of wild-type (wt) S100A9 by alanine (S100A9(N69A), S100A9(E78A) and S100A9(N69A+E78A)). Thus, these mutants lack essential ligands for calcium-coordination.23

First we found that the formation of S100A8/S100A9 heterodimers is unaffected by the different mutations. On the other hand, we could show that the EF-hand mutations prevent the formation of (S100A8/S100A9)2 heterotetramer complexes in the presence of calcium. Furthermore, lack of calcium-dependent tetramer formation of the mutants has functional consequences. While the wt complex promotes tubulin polymerization in the presence of calcium, the mutant S100A8/S100A9 complexes almost completely lack polymerization activity.

Thus, we demonstrate for the first time for a member of the S100 protein family that the calcium-dependent formation of higher-order oligomers is a prerequisite for physiological function(s) of these proteins.

Section snippets

Formation of S100A8/S100A9 heterodimers

The yeast two-hybrid technology was chosen as an in vivo system to characterize the heterodimerization properties of the S100A9 EF-hand mutants N69A, E78A and N69A+E78A in combination with wt S100A8. As shown in Figure 1(a), all S100A9 pAS2-1 constructs interact with S100A8 pACT2, indicating that the formation of S100A8/S100A9 heterodimers is unaffected by the mutations in the C-terminal EF-hand of S100A9. To confirm these results by an independent method, we used an in vitro protein–protein

Discussion

The S100 family is the largest group of calcium-binding EF-hand proteins. Family members are characterized by a tissue-specific expression pattern and by diverse functional roles in cell differentiation, migration and apoptosis.4, 5 S100A8 and S100A9 are the major calcium-binding proteins in neutrophils and monocytes.1, 25 Both proteins play a role in calcium-induced signaling where they are involved in the organization of cytoskeletal structures during transendothelial migration of activated

Yeast two-hybrid system

Human S100A8 and S100A9 wt cDNAs were cloned in-frame to either the GAL4 DNA-binding domain of the pAS2-1 vector [5′-NdeI; 3′-BamHI] or the GAL4 activation domain of the pACT2 vector [5′-NcoI; 3′-BamHI] (Clontech, Heidelberg, Germany). Construction of the S100A9 point mutants was achieved with the QuickChange™ Site-directed Mutagenesis Kit (Stratagene, Heidelberg, Germany). Yeast transformation was performed by the high-efficiency lithium acetate method as described by Propper et al.18

Acknowledgements

We thank U. Keller and H. Nüsse (Institute for Medical Physics and Biophysics) for excellent technical assistance. This work was supported by grants from the IZKF Muenster (Ro2/012/06) and the “Sonderforschungsbereich” 293.

References (41)

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    S100 proteins fold into a four-helix globular domain that is sometimes followed by a C-terminal, unstructured extension of variable length (Donato et al., 2013). The minimal functional unit is a homo- or heterodimer, but higher order oligomers are also encountered (Leukert et al., 2006). As for other S100 proteins, S100A9 active conformation requires the presence of two Ca2+ ions, one in each EF-hand motif, for proper effector binding (Vogl et al., 2006).

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N.L. and T.V. contributed equally to this work.

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