Department of Chemistry and Biochemistry and the University of California, Los Angeles-US Department of Energy Institute of Genomics and Proteomics, University of California, Los Angeles, CA 90095.
Division of Oral Biology and Medicine, University of California, Los Angeles, CA 90095.
Proc Natl Acad Sci U S A. 2021 Mar 23;118(12). doi: 10.1073/pnas.2019649118.
Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine-isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.
革兰氏阳性菌在其表面组装菌毛（纤毛）以黏附宿主组织并促进微生物的相互作用。这些毛发状结构虽然非常细（1 至 5nm），但具有令人印象深刻的拉伸强度，因为其蛋白质成分（菌毛蛋白）通过菌毛特异性天冬酰胺内肽酶通过赖氨酸异肽键共价交联在一起。虽然已经确定了分离的菌毛蛋白的原子结构，但天冬酰胺内肽酶如何将它们连接在一起以及这些菌毛内交联如何稳定菌毛结构尚不清楚。使用重建的菌毛组装系统和混合结构生物学方法，我们阐明了交联界面的溶液结构和动力学，该交联界面重复构建来自 的典型 SpaA 菌毛。我们表明，天冬酰胺内肽酶催化的相邻 SpaA 菌毛之间 K190-T494 异肽键的引入导致它们形成刚性界面，其中 LPLTG 分拣信号插入到大结合槽中。对交联反应的细胞和定量动力学测量揭示了菌毛生物发生的机制。我们提出， 中的菌毛特异性天冬酰胺内肽酶使用闩锁机制选择 SpaA 上的 K190 进行交联，其中分拣信号部分从酶转移到 SpaA 中的一个结合槽中，以促进催化。SpaA 中的保守环促进了这个过程，交联后形成一个稳定的闩锁，覆盖 K190-T494 异肽键。SpaA 菌毛的结构和天冬酰胺内肽酶催化组装机制的一般特征可能在革兰氏阳性菌中保守。
