Lira-Navarrete Erandi, de Las Rivas Matilde, Compañón Ismael, Pallarés María Carmen, Kong Yun, Iglesias-Fernández Javier, Bernardes Gonçalo J L, Peregrina Jesús M, Rovira Carme, Bernadó Pau, Bruscolini Pierpaolo, Clausen Henrik, Lostao Anabel, Corzana Francisco, Hurtado-Guerrero Ramon
BIFI, University of Zaragoza, BIFI-IQFR (CSIC) Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, Zaragoza 50018, Spain.
Departamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química, E-26006 Logroño, Spain.
Nat Commun. 2015 May 5;6:6937. doi: 10.1038/ncomms7937.
Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.
蛋白质O-糖基化由多肽N-乙酰半乳糖胺转移酶（GalNAc-Ts）控制，这些酶独特地具有催化结构域和凝集素结构域。GalNAc-Ts的凝集素结构域如何促成糖肽特异性和催化作用的潜在分子基础仍不清楚。在此，我们展示了GalNAc-T2与糖肽复合物的首个晶体结构，结合增强采样分子动力学模拟，证明了一种协同机制，即凝集素结构域使糖肽的游离受体位点能够结合到催化结构域中。原子力显微镜和小角X射线散射实验进一步揭示了GalNAc-T2的动态构象景观以及紧密结构在有效催化中所起的重要作用。我们的模型表明，GalNAc-T2的活性谱由构象异质性决定，并依赖于位于催化结构域和凝集素结构域之间的柔性连接子。我们的结果还阐明了GalNAc-Ts如何用O-聚糖对蛋白质进行密集修饰。
