C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C-mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound. The structures indicate how the C-mannosylation sequon is recognized by this CMT and its paralogs, and how sequon binding triggers conformational activation of the donor substrate: a process relevant to all glycosyltransferase C superfamily enzymes. Our structural data further indicate that the CMTs adopt an unprecedented electrophilic aromatic substitution mechanism to enable the C-glycosylation of proteins. These results afford opportunities for understanding human disease and therapeutic targeting of specific CMT paralogs.

C连接糖基化对于参与细胞通讯过程的分泌蛋白和跨膜蛋白的运输、折叠和功能至关重要。安装修饰的色氨酸C-甘露糖基转移酶 (CMT) 通过未知机制将甘露糖附着到新生多肽链中 WxxW/C 序列的第一个色氨酸上。在这里，我们报告了秀丽隐杆线虫CMT 在四种关键状态下的低温电子显微镜结构：apo、受体肽结合、供体-基质类似物结合以及作为肽和供体-基质模拟物结合的捕获三元复合物。这些结构表明C-甘露糖基化序列子如何被该 CMT 及其旁系同源物识别，以及序列子结合如何触发供体底物的构象激活：与所有糖基转移酶 C 超家族酶相关的过程。我们的结构数据进一步表明，CMT 采用前所未有的亲电芳香取代机制来实现蛋白质的 C-糖基化。这些结果为了解人类疾病和特定 CMT 旁系同源物的治疗靶向提供了机会。