Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methyl-methyl NOESY
Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methyl-methyl NOESY
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DOI：
10.1007/s10858-018-0183-4
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发表时间：
2018-04-01
影响因子：
2.7
通讯作者：
Peters, Thomas
中科院分区：
文献类型：
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作者：
Fluegge, Friedemann;Peters, Thomas
人血型 A 和 B 糖基转移酶（GTA、GTB）是高度同源的糖基转移酶。许多高分辨率晶体结构表明这些酶在底物结合后从开放构象转化为催化活性的闭合构象。然而，糖基转移的机制仍然存在争议，并且构象转变的确切性质和时间尺度尚不清楚。核磁共振提供了各种实验来进一步阐明这些未解决的问题。因此，第一步我们分配了 MILVA 标记的 GTA 和 GTB 样品中的所有甲基共振信号，这对于 70 kDa 同二聚体蛋白来说仍然是一项具有挑战性的任务。分配是通过甲基-甲基 NOESY 实验以及使用高分辨率晶体结构作为模板对镧系元素引起的赝接触位移 (PCS) 的测量获得的。由底物类似物结合引起的PCS和化学位移扰动表明，在存在镧系元素离子的情况下不采用完全闭合状态。
Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions. Therefore, in a first step we have assigned all methyl resonance signals in MILVA labeled samples of GTA and GTB, still a challenging task for 70 kDa homodimeric proteins. Assignments were obtained from methyl-methyl NOESY experiments, and from measurements of lanthanide-induced pseudocontact shifts (PCS) using high resolution crystal structures as templates. PCSs and chemical shift perturbations, induced by substrate analogue binding, suggest that the fully closed state is not adopted in the presence of lanthanide ions.