BACKGROUND The multi-domain protein InlB (internalin B) from Listeria monocytogenes is an agonist of the human receptor tyrosine kinase MET. Only the internalin domain directly interacts with MET. The internalin domain consists of seven central leucine-rich repeats (LRRs) flanked by an N-terminal helical cap domain and a C-terminal immunoglobulin-like structure. A potential function of the N-terminal cap in receptor binding could so far not be demonstrated by deleting the cap, since the cap is also implicated in nucleating folding of the LRR domain. RESULTS We generated an InlB variant (YopM-InlB) in which the InlB cap domain was replaced by the unrelated N-terminal capping structure of the LRR protein YopM from Yersinia enterocolitica. The crystal structure of the engineered protein shows that it folds properly. Because the first LRR is structurally closely linked to the cap domain, we exchanged LRR1 along with the cap domain. This resulted in unexpected structural changes extending to LRR2 and LRR3, which are deeply involved in MET binding. As a consequence, the binding of YopM-InlB to MET was substantially weaker than that of wild type InlB. The engineered protein was about one order of magnitude less active in colony scatter assays than wild type InlB. CONCLUSIONS We obtained a well-behaved InlB variant with an altered N-terminal capping structure through protein design. The reduced affinity for MET precludes a straightforward interpretation of the results from cell-based assays. Still, the engineered hybrid protein induced cell scatter, suggesting that the cap is required for folding and stability of InlB but is not essential for interactions that assemble the signalling-active receptor complex. The cap swap approach described here is clearly applicable to other L. monocytogenes internalins and other LRR proteins such as YopM and may yield useful structure/function correlates within this protein family.

中文翻译：

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工程化 YopM-InlB 杂合蛋白的晶体结构。

背景技术来自单核细胞增生李斯特菌的多结构域蛋白InlB(internalin B)是人受体酪氨酸激酶MET的激动剂。只有内部域直接与 MET 交互。internalin 结构域由 7 个中央富含亮氨酸的重复序列 (LRR) 组成，两侧是 N 端螺旋帽结构域和 C 端免疫球蛋白样结构。N-末端帽在受体结合中的潜在功能到目前为止还不能通过删除帽来证明，因为帽也与 LRR 结构域的成核折叠有关。结果 我们生成了一个 InlB 变体 (YopM-InlB)，其中 InlB 帽结构域被来自 Yersinia enterocolitica 的 LRR 蛋白 YopM 的不相关 N 端加帽结构所取代。工程蛋白的晶体结构表明它可以正确折叠。因为第一个 LRR 在结构上与帽域密切相关，我们将 LRR1 与帽域交换。这导致了延伸到 LRR2 和 LRR3 的意外结构变化，这些变化与 MET 结合密切相关。因此，YopM-InlB 与 MET 的结合明显弱于野生型 InlB。与野生型 InlB 相比，工程化蛋白质在菌落分散测定中的活性低约一个数量级。结论 我们通过蛋白质设计获得了具有改变的 N 末端加帽结构的表现良好的 InlB 变体。对 MET 的亲和力降低排除了对基于细胞的测定结果的直接解释。尽管如此，工程化的杂合蛋白诱导细胞分散，表明帽子是 InlB 折叠和稳定性所必需的，但对于组装信号活性受体复合物的相互作用不是必需的。此处描述的上限交换方法显然适用于其他单核细胞增生李斯特菌内蛋白和其他 LRR 蛋白，例如 YopM，并且可能在该蛋白家族中产生有用的结构/功能相关性。