Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. II (CE1428EHA), Buenos Aires, Argentina; Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN) CONICET. Ciudad Universitaria, Pab. II (CE1428EHA), Buenos Aires, Argentina.
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK; Dipartimento di Chimica e Farmacia, Università degli Studi di Sassari, Via Muroni 23A, 07100 Sassari, SS, Italy.
Structure. 2021 Apr 1;29(4):357-370.e9. doi: 10.1016/j.str.2020.11.017. Epub 2020 Dec 21.
UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name "Parodi limit" the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70-80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a "one-size-fits-all adjustable spanner" UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.
UDP-葡萄糖：糖蛋白糖基转移酶（UGGT）将错误折叠的糖蛋白标记为内质网保留。我们报告了全长嗜热毛壳菌 UGGT（CtUGGT）、两个 CtUGGT 双半胱氨酸突变体及其 TRXL2 结构域截断（CtUGGT-ΔTRXL2）的晶体结构。CtUGGT 分子动力学（MD）模拟捕捉到了扩展构象，并揭示了夹钳、弯曲和扭曲的域间运动。我们将“Parodi 极限”命名为在同一糖蛋白上，由于在该位点识别到错误折叠，单体 UGGT 在体外可以重新糖基化的错误折叠位点和 N-连接聚糖之间的最大距离。基于 MD 模拟，我们估计 Parodi 极限约为 70-80Å。糖蛋白晶体结构中糖蛋白残基与其最近的 N-连接糖基化位点之间距离的频率分布表明，Parodi 极限与 UGGT 在体内的活性有关。我们的数据支持 UGGT 底物识别的“一刀切的可调节扳手套件”模型，其中 UGGT TRXL2 结构域起着重要作用。
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