S100A12, an EF-hand calcium-binding protein, can be secreted by a variety of cell types and plays proinflammatory roles in a number of pathological conditions. Although S100A12 has been shown to interact with heparan sulfate (HS), the molecular detail of the interaction remains unclear. Here we investigate the structural basis of S100A12-HS interaction and how the interaction is regulated by the availability of divalent cations and the oligomeric states of S100A12. We discovered that S100A12-HS interaction requires calcium, while zinc can further enhance binding by inducing S100A12 hexamerization. In contrast, the apo form and zinc-induced tetramer form were unable to bind HS. Guided by the crystal structures of S100A12, we have identified the HS-binding site of S100A12 by site-directed mutagenesis. Characterization of the HS-binding site of S100A12 allowed us to convert the non-HS-binding apo and tetramer forms of S100A12 into a high affinity HS-binding variant by engineering a single point mutation. Using a HS oligosaccharide microarray we demonstrated that the N43K mutant displayed markedly enhanced selectivity towards longer HS oligosaccharides compared to the WT S100A12, likely due to the expanded dimension of the reengineered HS-binding site in the mutant. This unexpected finding strongly suggests that HS-binding sites of proteins might be amenable for engineering.

中文翻译：

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S100A12-硫酸乙酰肝素相互作用的表征和工程。

S100A12 是一种 EF 手钙结合蛋白，可以由多种细胞类型分泌，并在多种病理条件下发挥促炎作用。尽管 S100A12 已被证明与硫酸乙酰肝素 (HS) 相互作用，但相互作用的分子细节仍不清楚。在这里，我们研究了 S100A12-HS 相互作用的结构基础以及相互作用如何受二价阳离子的可用性和 S100A12 的寡聚状态调节。我们发现 S100A12-HS 相互作用需要钙，而锌可以通过诱导 S100A12 六聚化进一步增强结合。相比之下，apo 形式和锌诱导的四聚体形式无法结合 HS。以 S100A12 的晶体结构为指导，我们通过定点诱变确定了 S100A12 的 HS 结合位点。S100A12 的 HS 结合位点的表征使我们能够通过设计单点突变将 S100A12 的非 HS 结合 apo 和四聚体形式转化为高亲和力的 HS 结合变体。我们使用 HS 寡糖微阵列证明，与 WT S100A12 相比，N43K 突变体对更长的 HS 寡糖显示出显着增强的选择性，这可能是由于突变体中重新设计的 HS 结合位点的尺寸扩大。这一意外发现强烈表明蛋白质的 HS 结合位点可能适合进行工程改造。我们使用 HS 寡糖微阵列证明，与 WT S100A12 相比，N43K 突变体对更长的 HS 寡糖显示出显着增强的选择性，这可能是由于突变体中重新设计的 HS 结合位点的尺寸扩大。这一意外发现强烈表明蛋白质的 HS 结合位点可能适合进行工程改造。我们使用 HS 寡糖微阵列证明，与 WT S100A12 相比，N43K 突变体对更长的 HS 寡糖显示出显着增强的选择性，这可能是由于突变体中重新设计的 HS 结合位点的尺寸扩大。这一意外发现强烈表明蛋白质的 HS 结合位点可能适合进行工程改造。