以分子動態模擬探討鋅離子對c-Cbl RING domain結構穩定性的影響

在真核生物中，蛋白質的泛素化過程靠著泛素活化酶(E1)、泛素轉移酶(E2)、以及泛素連接酶(E3)扮演著調節多樣性生化程序的重要角色。其中，泛素連接酶是賦予泛素化專一性以及將泛素轉移至特殊待降解的目標蛋白上的重要關鍵之ㄧ。RING型泛素連接酶上的RING domain是需要鋅離子輔助形成其三級摺疊的小型結構蛋白。於本研究中，我們利用c-Cbl RING domain在不同條件下的分子動態模擬來詳細呈現其結構完整性與鋅離子之間的關係。模擬結果顯示鋅離子確實對維持c-Cbl RING domain的三級與二級結構穩定具有相當重要的貢獻。模擬結果進一步展現c-Cbl RING domain二級結構的穩定性是由金屬離子螯合點內或附近的氫鍵網絡所決定的。我們也論證鋅離子螯合在第二個金屬螯合點對於提升c-Cbl RING domain結構的完整性的貢獻比第一個金屬離子螯合點更多。接著進一步研究c-Cbl、CNOT4、以及p44的RING domain於模擬過程中的結構特色，我們發現p44上的recognition site的穩定性明顯低於c-Cbl以及CNOT4，而整體三級與二級結構穩定性卻無顯著差異。除此之外，c-Cbl以及CNOT4的兩個金屬螯合點之間的結構特性也分別與p44明顯不同。由此，我們推論具有E3功能的RING domain必須具有一個高度穩定的recognition site。雖然仍需要更多證據來證實我們的推論，不過本研究結果與前人實驗結果仍呈現良好的一致性。

關鍵字

泛素化； E1 ； E2 ； E3 ； RING domain ；鋅離子； c-Cbl domain ；金屬離子螯合點

並列摘要

In eukaryotic cells, ubiquitylation of proteins plays a critical role in regulating diverse cell processes by the ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin protein ligase (E3). The E3 enzymes are the key component that confers specificity to the ubiquitylation process and directs the conjugation of ubiquitin to the specific target protein. The RING domains are small structured protein domains that require the coordination of zinc ions for a stable tertiary fold and some of them are involved in RING E3s. In this study, we reported detailed relationships between the structural integrity of the c-Cbl RING domain and the two zinc ions under various conditions by molecular dynamics simulations. Our results showed that these two zinc ions indeed play an important role in maintaining the stability of both secondary and tertiary structures of the c-Cbl RING domain. Furthermore, our results also reveal that the secondary structural stabilities of the c-Cbl RING domain are mainly determined by the hydrogen bond networks in or near the stable metal ion binding site. Our results also demonstrate that the zinc ion binding site 2 (S2) contributes more significantly on the structural integrity of c-Cbl RING domain comparing to S1. Sequentially, we investigated the structural features of the RING domains of c-Cbl, CNOT4, and p44 using MD simulations. Several interesting features were observed. For example, the stability of recognition site of p44 is greatly lower than of that of c-Cbl and CNOT4. However, there are no significant differences between the tertiary or secondary structural stability of p44, c-Cbl, and CNOT4. Moreover, the structural features of the two zinc ion binding sites of p44 are different from those of c-Cbl, and CNOT4. Therefore, we suggested that the recognition site with high structural stability is a fundamental requirement for a RING domain to function as E3 ligases. Although more experimental evidences are required to reinforce our hypothesis, our results are in good agreement with the results from the previous experimental and computational studies.

並列關鍵字

ubiquitylation ； E1 ； E2 ； E3 ； RING domain ； zinc ion ； c-Cbl domain ； metal ion binding site

參考文獻

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以分子動態模擬探討鋅離子對c-Cbl RING domain結構穩定性的影響

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