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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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.