Abstract：
Staphylococcus aureus expresses three high-affinity neutrophil serine protease (NSP) inhibitors known as the extracellular adherence protein domain (EAPs) proteins. Whereas EapH1 and EapH2 are comprised of a single EAP domain, the modular extracellular adherence protein (Eap) from S. aureus strain Mu50 consists of four EAP domains. We recently reported that EapH2 can simultaneously bind and inhibit cathepsin-G (CG) and neutrophil elastase (NE), which are the two most abundant NSPs. This unusual property of EapH2 arises from independent CG and NE-binding sites that lie on opposing faces of its EAP domain. Here we used X-ray crystallography and enzyme assays to show that all four individual domains of Eap (i.e. Eap1, Eap2, Eap3, and Eap4) exhibit an EapH2-like ability to form ternary complexes with CG and NE that inhibit both enzymes simultaneously. We found that Eap1, Eap2, and Eap3 have similar functional profiles insofar as NSP inhibition is concerned but that Eap4 displays an unexpected ability to inhibit two NE enzymes simultaneously. Using X-ray crystallography, we determined that this second NE-binding site in Eap4 arises through the same region of its EAP domain that also comprises its CG-binding site. Interestingly, small angle X-ray scattering data showed that stable tail-to-tail dimers of the NE/Eap4/NE ternary complex exist in solution. This arrangement is compatible with NSP-binding at all available sites in a two-domain fragment of Eap. Together, our work implies that Eap is a polyvalent inhibitor of NSPs. It also raises the possibility that higher-order structures of NSP-bound Eap may have unique functional properties.
摘要：
金黄色葡萄球菌表达三种高亲和力中性粒细胞丝氨酸蛋白酶（NSP）抑制剂，称为胞外粘附蛋白结构域（EAP）蛋白。而EapH1和EapH2由单个EAP域组成，金黄色葡萄球菌菌株Mu50的模块化细胞外粘附蛋白（Eap）由四个EAP结构域组成。我们最近报道EapH2可以同时结合和抑制组织蛋白酶G（CG）和中性粒细胞弹性蛋白酶（NE），这是两个最丰富的NSP。EapH2的这种不寻常特性源于位于其EAP结构域相对面上的独立CG和NE结合位点。在这里,我们使用X射线晶体学和酶测定来显示Eap的所有四个单独结构域(即Eap1、Eap2、Eap3和Eap4)表现出与CG和NE形成三元复合物的EapH2样能力,其同时抑制两种酶。我们发现Eap1，Eap2和Eap3在NSP抑制方面具有相似的功能谱，但是Eap4表现出意想不到的同时抑制两种NE酶的能力。用X射线晶体学,我们确定Eap4中的第二个NE结合位点通过其EAP结构域的相同区域产生,该区域也包含其CG结合位点.有趣的是,小角度X射线散射数据表明，溶液中存在NE/Eap4/NE三元配合物的稳定的尾对尾二聚体。这种安排与Eap的两结构域片段中所有可用位点的NSP结合相容。一起,我们的工作表明Eap是NSPs的多价抑制剂。这也增加了NSP结合的Eap的高阶结构可能具有独特功能特性的可能性。
