Levin Mikhail K, Gurjar Madhura M, Patel Smita S
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2003 Jun 27;278(26):23311-6. doi: 10.1074/jbc.M301283200. Epub 2003 Mar 26.
The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the beta-gamma methylene/amine analogs of ATP, ATPgammaS, or [AlF4]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF3]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3' single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length.
丙型肝炎病毒（HCV）的解旋酶利用ATP水解产生的能量解开核酸。ATP酶循环被认为会诱导蛋白质构象变化，以驱动解旋酶沿核酸长度进行易位。我们研究了HCV解旋酶与核酸结合的能量学，以了解解旋酶的核苷酸连接状态如何决定其核酸结合位点的构象。由于解旋酶的大多数核苷酸连接状态因快速的ATP水解而短暂存在，因此分析了几种化合物以找到一种有效的不可水解的ATP类似物。我们发现，ATP的β-γ亚甲基/胺类似物、ATPγS或[AlF4]ADP在抑制HCV解旋酶的ATP酶活性方面无效。另一方面，发现[BeF3]ADP是ATP酶活性的有效抑制剂，并且它以1:1的化学计量比紧密结合到HCV解旋酶上。平衡结合研究表明，在没有ATP或存在ADP的情况下，HCV解旋酶以高亲和力结合单链核酸。与ATP类似物结合后，观察到对单链DNA的亲和力降低了100倍。在具有3'单链尾巴的双链DNA和RNA中也观察到了亲和力的降低，但在双链DNA中未观察到。这项研究的结果表明，HCV解旋酶的核酸结合位点受到ATP酶反应的变构调节。ATP的结合能用于使HCV解旋酶从紧密结合状态中释放出来，以促进易位。而ATP水解和产物释放步骤则促进解旋酶与核酸的紧密重新结合。基于这些结果，我们提出了一个布朗运动模型，用于解释HCV解旋酶沿核酸长度的单向易位。
