Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-CoV-2. Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) protein, enabling virus–host membrane fusion and infection of the airways. We present here a recombinant production strategy for enzymatically active TMPRSS2 and characterization of its matured proteolytic activity, as well as its 1.95 Å X-ray cocrystal structure with the synthetic protease inhibitor nafamostat. Our study provides a structural basis for the potent but nonspecific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that explain specificity. TMPRSS2 cleaved SARS-CoV-2 S protein at multiple sites, including the canonical S1/S2 cleavage site. We ranked the potency of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.4 nM to 120 µM and determined inhibitor mechanisms of action, providing the groundwork for drug development efforts to selectively inhibit TMPRSS2.

跨膜蛋白酶丝氨酸 2 (TMPRSS2) 已被确定为病毒进入和 SARS-CoV-2 发病机制的关键宿主细胞因子。具体而言，TMPRSS2 蛋白水解处理 SARS-CoV-2 刺突 (S) 蛋白，从而实现病毒-宿主膜融合和气道感染。我们在这里展示了一种用于酶促活性 TMPRSS2 的重组生产策略及其成熟蛋白水解活性的表征，以及其与合成蛋白酶抑制剂萘莫司他的 1.95 Å X 射线共晶结构。我们的研究为萘莫司他的有效但非特异性抑制提供了结构基础，并确定了解释特异性的 TMPRSS2 底物结合口袋的显着特征。TMPRSS2 在多个位点切割 SARS-CoV-2 S 蛋白，包括典型的 S1/S2 切割位点。