Chain-Selective Isotopic Labeling of the Heterodimeric Type III Secretion Chaperone, Scc4:Scc1, Reveals the Total Structural Rearrangement of the Chlamydia trachomatis Bi-Functional Protein, Scc4.
Chain-Selective Isotopic Labeling of the Heterodimeric Type III Secretion Chaperone, Scc4:Scc1, Reveals the Total Structural Rearrangement of the Chlamydia trachomatis Bi-Functional Protein, Scc4.
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DOI：
10.3390/biom10111480
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发表时间：
2020-10-24
期刊：
影响因子：
5.5
通讯作者：
Macnaughtan MA
中科院分区：
文献类型：
--
作者：
Ukwaththage TO;Keane SM;Shen L;Macnaughtan MA
Scc 4是一种来自沙眼衣原体（CT）的不寻常的双功能蛋白，其功能为III型分泌系统（T3 SS）伴侣蛋白和RNA聚合酶（RNAP）结合蛋白。这两种功能都需要在CT发育周期的特定阶段与蛋白质伴侣相互作用。作为T3 SS分子伴侣，Scc 4在发育后期结合Scc 1形成异二聚体复合物，其分子伴侣是重要的毒力效应子CopN。在发育的早中期，Scc 4通过结合含σ66的RNAP全酶来调节T3 SS基因的表达。为了研究的结构和缔合机制的Scc 4：Scc 1 T3 SS分子伴侣复合物使用核磁共振（NMR）光谱，我们开发了一种方法来选择性地标记的Scc 4：Scc 1复合物的每个链与15 N-同位素。该方法允许一种蛋白质在NMR光谱中一次可见，这大大减少了共振重叠，并允许比较游离和结合Scc 4的骨架结构。15 N-Scc 4：Scc 1和Scc 4：15 N-Scc 1复合物的1H，15 N-杂原子单量子相干光谱分别显示了Scc 4在结合Scc 1时的总结构重排和与Scc 1分离的动态区域。链选择性标记方法的发展揭示了Scc 4和Scc 1的缔合需要Scc 1的部分变性以形成高亲和力复合物，而在非变性条件下分离的蛋白质之间发生低亲和力相互作用。这些结果为Scc 4的功能转换机制和Scc 4：Scc 1在CT中的相互作用提供了新的模型。
Scc4 is an unusual bi-functional protein from Chlamydia trachomatis (CT) that functions as a type III secretion system (T3SS) chaperone and an RNA polymerase (RNAP)-binding protein. Both functions require interactions with protein partners during specific stages of the CT developmental cycle. As a T3SS chaperone, Scc4 binds Scc1 during the late stage of development to form a heterodimer complex, which chaperones the essential virulence effector, CopN. During the early-middle stage of development, Scc4 regulates T3SS gene expression by binding the σ66-containing RNAP holoenzyme. In order to study the structure and association mechanism of the Scc4:Scc1 T3SS chaperone complex using nuclear magnetic resonance (NMR) spectroscopy, we developed an approach to selectively label each chain of the Scc4:Scc1 complex with the 15N-isotope. The approach allowed one protein to be visible in the NMR spectrum at a time, which greatly reduced resonance overlap and permitted comparison of the backbone structures of free and bound Scc4. 1H,15N-heteronuclear single quantum coherence spectra of the 15N-Scc4:Scc1 and Scc4:15N-Scc1 complexes showed a total structural rearrangement of Scc4 upon binding Scc1 and a dynamic region isolated to Scc1, respectively. Development of the chain-selective labeling approach revealed that the association of Scc4 and Scc1 requires partial denaturation of Scc1 to form the high affinity complex, while low affinity interactions occurred between the isolated proteins under non-denaturing conditions. These results provide new models for Scc4′s functional switching mechanism and Scc4:Scc1 association in CT.
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