Substrate Profiling and High Resolution Co-complex Crystal Structure of a Secreted C11 Protease Conserved across Commensal Bacteria

Cysteine
proteases are among the most abundant hydrolytic enzymes
produced by bacteria, and this diverse family of proteins have significant
biological roles in bacterial viability and environmental interactions.
Members of the clostripain-like (C11) family of cysteine proteases
from commensal gut bacterial strains have recently been shown to mediate
immune responses by inducing neutrophil phagocytosis and activating
bacterial pathogenic toxins. Development of substrates, inhibitors,
and probes that target C11 proteases from enteric bacteria will help
to establish the role of these proteins at the interface of the host
and microbiome in health and disease. We employed a mass spectrometry-based
substrate profiling method to identify an optimal peptide substrate
of PmC11, a C11 protease secreted by the commensal bacterium Parabacteroides merdae. Using this substrate sequence information,
we synthesized a panel of fluorogenic substrates to calculate kcat and KM and to
evaluate the importance of the P2 amino acid for substrate turnover.
A potent and irreversible tetrapeptide inhibitor with a C-terminal
acyloxymethyl ketone warhead, Ac-VLTK-AOMK, was then
synthesized. We determined the crystal structure of PmC11 in complex
with this inhibitor and uncovered key active-site interactions that
govern PmC11 substrate recognition and specificity. This is the first
C11 protease structure in complex with a substrate mimetic and is
also the highest resolution crystal structure of a C11 protease to
date at 1.12 Å resolution. Importantly, subjecting human epithelial
cell lysates to PmC11 hydrolysis in combination with subtiligase-based
N-terminal labeling and tandem mass spectrometry proteomics complemented
the stringent substrate specificity observed in the in vitro substrate profiling experiment. The combination of chemical biological,
biophysical, and biochemical techniques presented here to elucidate
and characterize PmC11 substrate selectivity can be expanded to other
proteases and the development of chemical tools to study these essential
proteins in biologically relevant samples, such as the highly complex
distal gut microbiome.

半胱氨酸蛋白酶（Cysteine proteases）是细菌产生的丰度最高的水解酶类之一，这类结构多样的蛋白质家族在细菌存活与环境互作中发挥着重要生物学功能。来自共生肠道细菌菌株的梭菌蛋白酶样（C11）半胱氨酸蛋白酶家族成员，近期被证实可通过诱导中性粒细胞吞噬作用、激活细菌致病毒素来介导免疫应答。开发靶向肠道细菌C11蛋白酶的底物、抑制剂与探针，将有助于阐明这些蛋白质在宿主与微生物组界面的健康与疾病状态下的作用。我们采用基于质谱的底物谱分析方法，鉴定出了PmC11的最优肽底物——PmC11是共生细菌副杆菌Parabacteroides merdae分泌的一种C11蛋白酶。依托该底物序列信息，我们合成了一组荧光底物，用于计算催化常数（kcat）与米氏常数（KM），并评估P2位氨基酸对底物周转的重要性。随后我们合成了一种强效不可逆四肽抑制剂，其C端携带酰氧甲基酮弹头Ac-VLTK-AOMK。我们解析了该抑制剂与PmC11结合的晶体结构，揭示了调控PmC11底物识别与特异性的关键活性位点相互作用。这是首个结合底物模拟物的C11蛋白酶晶体结构，同时也是目前分辨率最高的C11蛋白酶晶体结构，分辨率达1.12 Å。值得注意的是，将人类上皮细胞裂解液经PmC11水解后，结合基于枯草杆菌蛋白酶的N端标记与串联质谱蛋白质组学分析，验证了体外底物谱实验中观察到的严格底物特异性。本文所采用的化学生物学、生物物理学与生物化学技术，可用于解析与表征PmC11的底物选择性，该方法可拓展至其他蛋白酶的研究，以及开发化学工具以在生物相关样本（如高度复杂的远端肠道微生物组）中研究这些必需蛋白质。