Extracellular adherence protein domain (EAP) proteins are high-affinity, selective inhibitors of neutrophil serine proteases (NSP), including cathepsin-G (CG) and neutrophil elastase (NE). Most Staphylococcus aureus isolates encode for two EAPs, EapH1 and EapH2, that contain a single functional domain and share 43% identity with one another. Although structure/function investigations from our group have shown that EapH1 uses a globally similar binding mode to inhibit CG and NE, NSP inhibition by EapH2 is incompletely understood due to a lack of NSP/EapH2 cocrystal structures. To address this limitation, we further studied NSP inhibition by EapH2 in comparison with EapH1. Like its effects on NE, we found that EapH2 is a reversible, time-dependent, and low nanomolar affinity inhibitor of CG. We characterized an EapH2 mutant which suggested that the CG binding mode of EapH2 is comparable to EapH1. To test this directly, we used NMR chemical shift perturbation to study EapH1 and EapH2 binding to CG and NE in solution. Although we found that overlapping regions of EapH1 and EapH2 were involved in CG binding, we found that altogether distinct regions of EapH1 and EapH2 experienced changes upon binding to NE. An important implication of this observation is that EapH2 might be capable of binding and inhibiting CG and NE simultaneously. We confirmed this unexpected feature by solving crystal structures of the CG/EapH2/NE complex and demonstrating their functional relevance through enzyme inhibition assays. Together, our work defines a new mechanism of simultaneous inhibition of two serine proteases by a single EAP protein.

细胞外粘附蛋白结构域 (EAP) 蛋白是中性粒细胞丝氨酸蛋白酶 (NSP) 的高亲和力、选择性抑制剂，包括组织蛋白酶-G (CG) 和中性粒细胞弹性蛋白酶 (NE)。大多数金黄色葡萄球菌分离株编码两种 EAP：EapH1 和 EapH2，它们包含单个功能域，并且彼此之间有 43% 的同一性。尽管我们小组的结构/功能研究表明，EapH1 使用全局相似的结合模式来抑制 CG 和 NE，但由于缺乏 NSP/EapH2 共晶结构，EapH2 对 NSP 的抑制作用尚不完全清楚。为了解决这一局限性，我们进一步研究了 EapH2 与 EapH1 的 NSP 抑制作用。与它对 NE 的作用一样，我们发现 EapH2 是一种可逆的、时间依赖性的、低纳摩尔亲和力的 CG 抑制剂。我们对 EapH2 突变体进行了表征，这表明 EapH2 的 CG 结合模式与 EapH1 相当。为了直接测试这一点，我们使用 NMR 化学位移微扰来研究 EapH1 和 EapH2 与溶液中 CG 和 NE 的结合。尽管我们发现 EapH1 和 EapH2 的重叠区域参与 CG 结合，但我们发现 EapH1 和 EapH2 的完全不同区域在与 NE 结合后经历了变化。这一观察结果的一个重要含义是 EapH2 可能能够同时结合和抑制 CG 和 NE。我们通过解析 CG/EapH2/NE 复合物的晶体结构并通过酶抑制测定证明其功能相关性，证实了这一意想不到的特征。我们的工作共同定义了一种通过单个 EAP 蛋白同时抑制两种丝氨酸蛋白酶的新机制。