Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250' and 280s Loops.
Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250' and 280s Loops.
复制标题
DOI：
10.1021/acs.biochem.2c00671
复制
发表时间：
2023-04-18
期刊：
影响因子：
2.9
通讯作者：
Phillips, Jonathan J.
中科院分区：
文献类型：
--
作者：
Kish, Monika;Subramanian, Sivaraman;Smith, Victoria;Lethbridge, Natasha;Cole, Lindsay;Vollmer, Frank;Bond, Nicholas. J.;Phillips, Jonathan J.
变构是蛋白质活化的基本机制，然而作为变构酶（例如糖原磷酸化酶（GlyP））的功能调节的基础的精确动态变化仍然知之甚少。尽管是第一个被描述的变构酶，但其结构调控仍然是一个具有挑战性的问题：GlyP活性位点（250′和280 s）的关键调控环稳定性较弱，经常丢失密度或在结构模型中具有较大的b因子。这导致了一个长期存在的假设，即GlyP调节是通过（无序）转换门控活性位点实现的，如Barford和约翰逊首先提出的。然而，测试这一点需要定量测量弱稳定的局部结构，迄今为止，在这样一个大的蛋白质中，这在技术上具有挑战性。氢氘交换质谱（HDX-MS）是研究蛋白质动力学的有力工具，毫秒HDX-MS具有测量弱稳定结构中的位点局部稳定性差异的能力，使得其对于研究GlyP中的变构调节特别有价值。在这里，我们使用毫秒HDX-MS测量糖原磷酸化酶B（GlyPb），磷酸化的活性形式（GlyPa），和抑制葡萄糖-6磷酸复合物（GlyPb：G6 P）在近氨基酸分辨率的局部结构扰动。我们的研究结果支持Barford和约翰逊假说的GlyP调节的关键调控环的动态变化提供了洞察。
Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250′ and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson. However, testing this requires a quantitative measurement of weakly stable local structure which, to date, has been technically challenging in such a large protein. Hydrogen–deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for studying protein dynamics, and millisecond HDX-MS has the ability to measure site-localized stability differences in weakly stable structures, making it particularly valuable for investigating allosteric regulation in GlyP. Here, we used millisecond HDX-MS to measure the local structural perturbations of glycogen phosphorylase b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution. Our results support the Barford and Johnson hypothesis for GlyP regulation by providing insight into the dynamic changes of the key regulatory loops.
登录
查看更多内容
影响因子：
--
作者：
Mueller M;Nidetzky B
通讯作者：
Nidetzky B
影响因子：
5.5
作者：
Beveridge R;Calabrese AN
通讯作者：
Calabrese AN
影响因子：
7.4
作者：
Kish, Monika;Smith, Victoria;Lethbridge, Natasha;Cole, Lindsay;Bond, Nicholas. J.;Phillips, Jonathan J.
通讯作者：
Phillips, Jonathan J.
DOI：
10.1073/pnas.1717621115
发表时间：
2018-03-20
影响因子：
11.1
作者：
Fan, Yifei;Cross, Penelope J.;Parker, Emily J.
通讯作者：
Parker, Emily J.
影响因子：
2.9
作者：
Al-Naqshabandi, Mohammed A.;Weis, David D.
通讯作者：
Weis, David D.