Zn2+ binding properties of single-point mutants of the C-terminal zinc finger of the HIV-1 nucleocapsid protein:: Evidence of a critical role of cysteine 49 in Zn2+ dissociation
Zn2+ binding properties of single-point mutants of the C-terminal zinc finger of the HIV-1 nucleocapsid protein:: Evidence of a critical role of cysteine 49 in Zn2+ dissociation
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DOI：
10.1021/bi015956g
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发表时间：
2002-04-02
期刊：
影响因子：
2.9
通讯作者：
Mély, Y
中科院分区：
文献类型：
--
作者：
Bombarda, E;Cherradi, H;Mély, Y
HIV-1核衣壳蛋白NCp 7的两个高度保守的Zn 2+指基序通过一个His和三个Cys残基的配位而强烈结合Zn 2+。为了进一步分析这些残基的作用，我们研究了Zn 2+结合和酸碱性质的四个单点突变体的短肽对应的远端指状基序的NCp 7。在每个突变体中，一个Zn 2 +-配位残基被非配位残基取代。利用Co2+的光谱特性，我们首先确定四种突变体保留了通过四或五坐标几何结构结合金属阳离子的能力，其中空配体位置可能被水分子占据。此外，通过H-1 NMR发现其中His 44被Ala取代的突变脱辅基肽的三个Cys残基的pK（a）值与天然肽的pK（a）值相似，这表明突变不影响Zn 2（+-）配位残基的酸碱性质。以Trp 37的荧光作为内源性探针，监测Zn ~（2+）的结合。在pH 7.5时，四种突变体的表观Zn 2+结合常数（在1.6 × 10（8）和1.3 × 10（10）M-1之间）与天然肽的表观Zn 2+结合常数相比大幅降低，但与各种宿主Zn 2+结合蛋白的表观Zn 2+结合常数相似。因此，在体内一个Zn 2+配位残基突变后病毒感染性的丧失可能与Zn 2+结合的完全丧失无关。Zn 2+结合的pH依赖性表明配位残基逐步结合Zn 2+，并且给定残基的结合所提供的自由能可以由已经结合到Zn 2+的残基的熵贡献来调制。最后，发现全肽中Cys 49的pK（a）为5.0，该值比其它Zn 2+配位残基的pK（a）高至少0.7个单位。这意味着，Cys 49可以作为一个开关的Zn 2+解离的远端指基序的NCp 7，一个功能，可能有助于Cys 49亲电攻击的高敏感性。
The two highly conserved Zn2+ finger motifs of the HIV-1 nucleocapsid protein, NCp7, strongly bind Zn2+ through coordination of one His and three Cys residues. To further analyze the role of these residues, we investigated the Zn2+ binding and acid-base properties of four single-point mutants of a short peptide corresponding to the distal finger motif of NCp7. In each mutant, one Zn2+-coordinating residue is substituted with a noncoordinating one. Using the spectroscopic properties of Co2+, we first establish that the four mutants retain their ability to bind a metal cation through a four- or five-coordinate geometry with the vacant ligand position(s) presumably occupied by water molecule(s). Moreover, the pK(a) values of the three Cys residues of the mutant apopeptide where His44 is substituted with Ala are found by H-1 NMR to be similar to those of the native peptide, suggesting that the mutations do not affect the acid-base properties of the Zn2(+-)coordinating residues. The binding of Zn2+ was monitored by using the fluorescence of Trp37 as an intrinsic probe. At pH 7.5, the apparent Zn2+ binding constants (between 1.6 x 10(8) and 1.3 x 10(10) M-1) of the four mutants are strongly reduced compared to those of the native peptide but are similar to those of various host Zn2+ binding proteins. As a consequence, the loss of viral infectivity following the mutation of one Zn2+-coordinating residue in vivo may not be related to the total loss of Zn2+ binding. The pH dependence of Zn2+ binding indicates that the coordinating residues bind Zn2+ stepwise and that the free energy provided by the binding of a given residue may be modulated by the entropic contribution of the residues already bound to Zn2+. Finally, the pK(a) of Cys49 in the holopeptide is found to be 5.0, a value that is at least 0.7 unit higher than those for the other Zn2+-coordinating residues. This implies that Cys49 may act as a switch for Zn2+ dissociation in the distal finger motif of NCp7, a feature that may contribute to the high susceptibility of Cys49 to electrophilic attack.