登革熱病毒非結構蛋白 NS3 之腺苷三磷酸水解酶之研究-NS3 蛋白和
腺苷三磷酸及核醣核酸三者間之交互關係
The studies of the ATPase activity of Dengue virus nonstructural protein NS3-The interaction among NS3 protein, ATP and RNA.
中文摘要
登革熱病毒非結構蛋白 3 (NS3) C 端含有 RNA 解旋酶的保守性序列，具有 NTPase、RNA triphosphatase 及 RNA 解旋酶活性。本論文的目的是分析由大腸 桿菌表現及純化的登革熱病毒 NS3 C 端的蛋白 (簡稱 NS3H)的 ATPase 活性，並 藉定點突變蛋白活性的變化，探討 NS3H 蛋白水解 ATP 的作用機制以及 NS3H 蛋白、ATP 和 RNA 的關係。實驗結果發現 NS3H 蛋白水解 ATP 反應需要二價陽 離子的參與，如 Mg2+、Mn2+及 Ca2+才可啟動，其中以 Mg2+效果最佳。Mg2+
的效應會被 Mn2+抑制，顯示反應中 MnATP 複合物對 NS3H 蛋白的親和力不弱 於 MgATP。NS3H 蛋白的 ATPase 活性可分被 rU35 和 NaCl 激活，兩因子激活 NS3H ATPase 反應對 ATP 的 Km 改變不大 (92-120 μM)，僅會提增 ATP 的水解 速率。顯示了 NaCl 和 rU35 主要影響 ATP 進入 ATPase 活性區後的水解效能。再 者 rU35 的激活效果會被高濃度 (10mM 以上)的 NaCl 抑制，代表著高濃度的 NaCl 會降低 NS3H 和核酸間的離子作用力，使得 NS3H 不易與核酸結合，進而影響後 續 ATPase 活性激活的效果。NS3H 蛋白 RNA 解旋酶保守性區的 motif I 之 K199A T200A 置換突變和 motif II 的 D284A 置換，均致使 NS3H 蛋白失去水解 ATP 的 能力。而 motif II 的 D284E 置換突變蛋白，仍保有水解 ATP 的能力和受核酸激 活的特性。因此 motifs I 和 II 與 ATP 的結合和水解有關連。NS3H 蛋白 RNA 解 旋酶保守區 gatekeepers 的 M429 和 W488 分別被置換時，ATPase 活性均會受到 影響。M429A 突變蛋白 ATPase 活性仍可被核酸激活，此突變蛋白與 ATP 的 Km 值無論有否受 rU35 的激活，其 Km 值與 NS3H 相較差異不大。W488A 的 ATPase 活性極低而且會受到核酸的抑制，且此受核酸的抑制效應和 M429A 受核酸的激 活效應皆可被高濃度的 NaCl 所遮蔽，這表示 M429A 和 W488A 皆可與核酸結 合，且結合後的酵素活性會因此而產生變化。至此我們認為 NS3H 蛋白與核酸結 合後可能會誘導蛋白結構產生改變，在不影響 ATP 的 Km 情況下，此結構的改 變會提增 ATP 在活性區內的水解速率，而 gatekeeper 的氨基酸會影響蛋白與核 酸結合後的結構改變，進而影響後續 ATPase 的水解效果。
英文摘要
The C terminal domain of dengue virus nonstructural protein 3 (NS3) contains the conserved motifs present in several RNA helicases and has NTPase, RNA
triphosphotase, and RNA helicase activities. In this research, we expressed and
purified a His-tag fusion protein containing the C terminal domain of dengue virus NS3 (named as NS3H). In addition, we constructed and prepared the substitution mutants of NS3H. We studied the ATPase activity of the wild type and the mutant proteins, and examined the relationship among NS3H, ATP, and RNA. The ATPase activity required divalent cation to function. Mg2+ was more effective than Mn2+ and Ca2+ in catalyzing ATP hydrolysis, while Mn2+ strongly inhibited the stimulatory effect of Mg2+. Thus, MnATP appears to have a higher affinity to NS3H than that of MgATP. Both NaCl and rU35 (U homopolymer of 35 nt long) accelerated the rate of ATP hydrolysis, but they did not alter the Km to ATP (90-120 µM). The increase of NaCl concentration from 10 mM to 30 mM eliminated the stimulatory effect of rU35 on ATP hydrolysis, suggesting the interaction between rU35 and NS3H was mainly electrostatic. The K199A, T200A substitutions in the motif I as well as the D284A substitution in the motif II abolished the ATPase activity of NS3H, while the D284E substitution mutant possessed the RNA stimulated ATPase activity. These results reveal that the motifs I and II participate in ATP binding and ATP hydrolysis. M429 and W488 were the “gatekeepers” of the conserved RNA helicase domain. The M429A substitution mutant interacted with ATP and rU35 in a manner similar to the wild type protein, while it hydrolyzed ATP less efficiently than that of NS3H. The W488A substitution mutant had a very low ATPase activity. Instead of being
stimulated by rU35, the ATPase activity of this mutant was inhibited by rU35, and the inhibitory effect of rU35 was attenuated when the NaCl concentration increased. Thus, the substitution at each gatekeeper may not affect the RNA binding activity of NS3H.
In summary, the results of this study suggest that the binding of RNA to NS3H may induce the conformation rearrangement of NS3H that increases the ATP hydrolysis rate but does not alter the Km to ATP. The gatekeeper mutants may have defect in the conformation rearrangement process, as a result the ATPase activity is attenuated.
