Roncase Emily J, González-Páez Gonzalo E, Wolan Dennis W
Departments of Molecular Medicine and Integrative Structural and Computational Biology , The Scripps Research Institute , 10550 North Torrey Pines Road , La Jolla , California 92037 , United States.
Biochemistry. 2019 Apr 2;58(13):1728-1737. doi: 10.1021/acs.biochem.9b00098. Epub 2019 Mar 14.
Commensal bacteria secrete proteins and metabolites to influence host intestinal homeostasis, and proteases represent a significant constituent of the components at the host:microbiome interface. Here, we determined the structures of the two secreted C11 cysteine proteases encoded by the established gut commensal Bacteroides thetaiotaomicron. We employed mutational analysis to demonstrate the two proteases, termed "thetapain" and "iotapain", undergo in trans autoactivation after lysine and/or arginine residues, as observed for other C11 proteases. We determined the structures of the active forms of thetapain and iotapain in complex with irreversible peptide inhibitors, Ac-VLTK-AOMK and biotin-VLTK-AOMK, respectively. Structural comparisons revealed key active-site interactions important for peptide recognition are more extensive for thetapain; however, both proteases employ a glutamate residue to preferentially bind small polar residues at the P2 position. Our results will aid in the design of protease-specific probes to ultimately understand the biological role of C11 proteases in bacterial fitness, elucidate their host and/or microbial substrates, and interrogate their involvement in microbiome-related diseases.
共生细菌分泌蛋白质和代谢产物以影响宿主肠道稳态，而蛋白酶是宿主与微生物群界面成分的重要组成部分。在此，我们确定了已确立的肠道共生菌嗜热栖粪杆菌编码的两种分泌型C11半胱氨酸蛋白酶的结构。我们采用突变分析来证明这两种蛋白酶，即“θ蛋白酶”和“ι蛋白酶”，如其他C11蛋白酶一样，在赖氨酸和/或精氨酸残基后发生反式自激活。我们分别确定了与不可逆肽抑制剂Ac-VLTK-AOMK和生物素-VLTK-AOMK结合的θ蛋白酶和ι蛋白酶活性形式的结构。结构比较显示，对于肽识别重要的关键活性位点相互作用，θ蛋白酶更为广泛；然而，两种蛋白酶都利用谷氨酸残基优先结合P2位置的小极性残基。我们的结果将有助于设计蛋白酶特异性探针，以最终了解C11蛋白酶在细菌适应性中的生物学作用，阐明其宿主和/或微生物底物，并探究它们与微生物群相关疾病的关系。
