Molecular mechanism of the Zn2+-induced folding of the distal CCHC finger motif of the HIV-1 nucleocapsid protein
Molecular mechanism of the Zn2+-induced folding of the distal CCHC finger motif of the HIV-1 nucleocapsid protein
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DOI：
10.1529/biophysj.106.101378
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发表时间：
2007-07-01
影响因子：
3.4
通讯作者：
Mely, Yves
中科院分区：
文献类型：
--
作者：
Bombarda, Elisa;Grell, Ernst;Mely, Yves
HIV-1核衣壳蛋白NCp 7含有两个高度保守的CCHC锌指结构。Zn 2+的结合驱动NCp 7从未折叠到高度折叠的结构，这对其功能至关重要。利用Trp 37的内源荧光，采用停流技术研究了NCp 7远端指的折叠过程，并通过pH依赖性的Zn 2+缔合和解离动力学研究了NCp 7远端指的折叠过程。Zn 2+结合被发现涉及四个不同的路径与四个去质子化状态的。恩格尔。每个结合途径涉及中间复合物的快速形成，该中间复合物随后在限速步骤中重排并稳定。已确定的平衡和动力学速率常数的完整的Zn 2+结合过程。在中性pH下，Zn 2+驱动折叠的优先途径意味着Zn 2+结合到去质子化的Cys（36）和His（44）残基，处于双齿状态。恩格尔。然后以500 s（-1）的速率常数将所得中间体转化为更合适的折叠形式，可能是通过Zn 2+周围的肽骨架重排以优化结合几何形状。这种形式，然后迅速导致。最终天然复合物，通过Cys（39）和Cys（49）残基的去质子化和配位水分子的分子内取代。Zn 2+的解离也具有多步过程的特征，并且通过去质子化的Zn 2+结合双齿态以3 s（-1）的速率常数发生最快。由于它们在折叠中的关键作用，鉴定的中间体。这项研究的第一个时间可能构成HIV治疗的潜在靶点。
HIV-1 nucleocapsid protein, NCp7, contains two highly conserved CCHC zinc fingers. Binding of Zn2+ drives NCp7 from an unfolded to a highly folded structure that is critical for its functions. Using the intrinsic fluorescence of Trp 37, we investigated, by the stopped-flow technique, the folding of NCp7 distal finger through the pH dependence of its Zn2+ association and dissociation kinetics. Zn2+ binding was found to involve four different paths associated with the four deprotonated states of the. nger. Each binding path involves the rapid formation of an intermediate complex that is subsequently rearranged and stabilized in a rate- limiting step. The equilibrium and kinetic rate constants of the full Zn2+- binding process have been determined. At neutral pH, the preferential pathway for the Zn2+- driven folding implies Zn2+ binding to the deprotonated Cys(36) and His(44) residues, in the bidentate state of the. nger. The resulting intermediate is then converted with a rate constant of 500 s(-1) into a more suitably folded form, probably through a rearrangement of the peptide backbone around Zn2+ to optimize the binding geometry. This form then rapidly leads to the. nal native complex, through deprotonation of Cys(39) and Cys(49) residues and intramolecular substitution of coordinated water molecules. Zn2+ dissociation is also characterized by a multistep process and occurs fastest via the deprotonated Zn2+ bound bidentate state with a rate constant of 3 s(-1) . Due to their critical role in folding, the intermediates identified for the. rst time in this study may constitute potential targets for HIV therapy.