PDF(Pubmed)
Abstract：
Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.
摘要：
溶组织梭菌分泌的梭状芽孢杆菌是ClanCD半胱氨酸肽酶C11家族的创始成员，这是许多细菌分泌的一组重要的肽酶。梭菌蛋白酶是精氨酸特异性内肽酶。由于其作为半胱氨酸肽酶的功效，它广泛用于实验室设置。尽管其重要性，但梭菌痛的结构仍未解决。在这里，我们描述了使用微晶电子衍射（MicroED）以2.5µ分辨率确定的溶组织梭状芽孢杆菌的活性形式的第一个结构。从聚焦离子束铣削后的单个纳米晶体确定结构。梭状芽孢杆菌的结构显示出典型的ClanCDα/β/α夹心结构和活性位点的Cys231/His176催化二元结构。它有一个大的负电性底物结合袋，显示出其容纳大型和多样化底物的能力。在残基452和457之间形成的重链中的环对于底物结合是潜在重要的。总之,这一结果证明了MicroED的重要性，以确定大分子的未知结构，如梭菌痛，可以进一步用作研究底物结合和设计针对此类肽酶的潜在抑制剂的平台。
