From the Departments of Biochemistry and.
Medicinal Chemistry, University of Washington, Seattle, Washington 98195.
J Biol Chem. 2019 Jan 18;294(3):783-793. doi: 10.1074/jbc.RA118.004247. Epub 2018 Nov 20.
SspH/IpaH bacterial effector E3 ubiquitin (Ub) ligases, unrelated in sequence or structure to eukaryotic E3s, are utilized by a wide variety of Gram-negative bacteria during pathogenesis. These E3s function in a eukaryotic environment, utilize host cell E2 ubiquitin-conjugating enzymes of the Ube2D family, and target host proteins for ubiquitylation. Despite several crystal structures, details of Ube2D∼Ub binding and the mechanism of ubiquitin transfer are poorly understood. Here, we show that the catalytic E3 ligase domain of SspH1 can be divided into two subdomains: an N-terminal subdomain that harbors the active-site cysteine and a C-terminal subdomain containing the Ube2D∼Ub-binding site. SspH1 mutations designed to restrict subdomain motions show rapid formation of an E3∼Ub intermediate, but impaired Ub transfer to substrate. NMR experiments using paramagnetic spin labels reveal how SspH1 binds Ube2D∼Ub and targets the E2∼Ub active site. Unexpectedly, hydrogen/deuterium exchange MS shows that the E2∼Ub-binding region is dynamic but stabilized in the E3∼Ub intermediate. Our results support a model in which both subunits of an Ube2D∼Ub clamp onto a dynamic region of SspH1, promoting an E3 conformation poised for transthiolation. A conformational change is then required for Ub transfer from E3∼Ub to substrate.
SspH/IpaH 细菌效应物 E3 泛素（Ub）连接酶在序列或结构上与真核 E3 无关，在发病机制过程中被各种革兰氏阴性菌利用。这些 E3 在真核环境中发挥作用，利用宿主细胞 Ube2D 家族的 E2 泛素连接酶，并将宿主蛋白靶向泛素化。尽管已经有几个晶体结构，但 Ube2D∼Ub 结合的细节和泛素转移的机制仍知之甚少。在这里，我们表明 SspH1 的催化 E3 连接酶结构域可以分为两个亚结构域：一个含有活性位点半胱氨酸的 N 端亚结构域和一个包含 Ube2D∼Ub 结合位点的 C 端亚结构域。设计用于限制亚结构域运动的 SspH1 突变显示出快速形成 E3∼Ub 中间体，但阻碍了 Ub 向底物的转移。使用顺磁自旋标记的 NMR 实验揭示了 SspH1 如何结合 Ube2D∼Ub 并靶向 E2∼Ub 活性位点。出乎意料的是，氢/氘交换 MS 表明 E2∼Ub 结合区域是动态的，但在 E3∼Ub 中间体中稳定。我们的结果支持这样一种模型，即 Ube2D∼Ub 的两个亚基都结合到 SspH1 的一个动态区域，促进了一种准备进行转硫醇反应的 E3 构象。然后需要构象变化才能将 Ub 从 E3∼Ub 转移到底物上。
