Division of Oral & Systemic Health Sciences, School of Dentistry, University of California, Los Angeles, California, USA.
Department of Chemistry, University of California, Irvine, Irvine, California, USA.
J Biol Chem. 2024 Jun;300(6):107329. doi: 10.1016/j.jbc.2024.107329. Epub 2024 Apr 26.
The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.
革兰氏阳性菌菌毛的两相组装始于由特定菌毛的天冬酰胺酰基转移酶（sortase）催化的不同菌毛聚合，随后由管家天冬酰胺酰基转移酶介导聚合产物在细胞壁上的锚定。在口腔放线菌中，特定菌毛的天冬酰胺酰基转移酶 SrtC2 不仅聚合 FimA 菌毛组装带有 CafA 尖端的 2 型菌毛，还可以作为锚定天冬酰胺酰基转移酶，当管家天冬酰胺酰基转移酶 SrtA 失活时，将 FimA 聚合体和 SrtC1 催化的 FimP 聚合体（1 型菌毛）连接到肽聚糖上。迄今为止，尚未阐明决定 SrtC2 独特底物特异性和双重酶活性的结构-功能决定因素。在这里，我们展示了分辨率为 2.10-Å 的 SrtC2 晶体结构。SrtC2 含有典型的天冬酰胺酰基转移酶折叠和 C 类天冬酰胺酰基转移酶的盖子，以及 SrtC2 特有的其他特征。SrtC2 的结构、生化和突变分析表明，SrtC2 的扩展盖子调节其双重活性。具体来说，我们证明 SrtC2 的聚合活性仍然通过丙氨酸取代、部分缺失和用 SrtC1 盖子替换 SrtC2 盖子得以维持。引人注目的是，这些突变导致 CafA 的菌毛掺入显著减少，导致 CafA 介导的多微生物相互作用受损。在 srtA 突变体中，SrtC2 盖子的部分缺失减少了 FimP 聚合体的表面锚定，而盖子交换突变增强了这一过程，同时这两种突变都减少了 FimA 菌毛的表面锚定。显然，SrtC2 的扩展盖子以底物选择性的方式赋予该酶细胞壁锚定活性。
