Highly Sensitive Analysis of the Interaction between HIV-1 Gag and Phosphoinositide Derivatives Based on Surface Plasmon Resonance
Highly Sensitive Analysis of the Interaction between HIV-1 Gag and Phosphoinositide Derivatives Based on Surface Plasmon Resonance
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DOI：
10.1021/bi9019274
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发表时间：
2010-06-29
期刊：
影响因子：
2.9
通讯作者：
Fujita, Mikako
中科院分区：
文献类型：
--
作者：
Anraku, Kensaku;Fukuda, Ryota;Fujita, Mikako
人类免疫缺陷病毒1型（HIV-1）Gag蛋白是HIV颗粒的主要结构成分。Pr 55（Gag）蛋白定位于质膜启动病毒组装。最近的研究表明，D-肌磷脂酰肌醇（P1）4，5-二磷酸（P1（4，5）P2）调节Pr 55（Gag）的定位和组装。我们确定了Pr 55（Gag）或其N-末端MA结构域和各种磷酸肌醇衍生物之间的结合亲和力，使用高灵敏度的表面等离子体共振（SPR）传感器和生物素化肌醇磷酸。使用这种方法得到的平衡解离常数反映了不同的幅度的酰基基团为基础的和磷酸基团为基础的相互作用。Pr 55（Gag）与1，4，5-IP 3复合的解离常数（K-D）（具有二价磷酸基团并且没有脂质基团的肌醇）的K-D为2170 μ M，而二-C-8-P1的K-D为2170 μ M，（不含二价磷酸基团的含脂质肌醇）为186 μ M，二-C-8-P1（4，5）P2（具有脂质和二价磷酸基团的肌醇）为47.4 μ M。当这些磷酸肌醇与MA复合时，观察到亲和力的相同趋势。我们的研究结果表明，在Pr 550（Gag）/MA结合中，疏水酰基链的贡献大于带负电荷的肌醇磷酸。此外，测试的每种肌醇磷酸（缺乏脂质基团）显示出不同的Pr 55（Gag）结合亲和力，这取决于磷酸基团的位置和数量。然而，磷酸基团的位置和数量对MA结合亲和力没有影响。
Human immunodeficiency virus type 1 (HIV-1) Gag protein is the principal structural component of the HIV particle. Localization of the Pr55(Gag) protein to the plasma membrane initiates virus assembly. Recent studies indicated that D-myo-phosphatidylinositol (P1) 4,5-bisphosphate (P1(4,5)P2) regulates Pr55(Gag) localization and assembly. We determined the binding affinity between Pr55(Gag) or its N-terminal MA domain and various phosphoinositide derivatives using a highly sensitive surface plasmon resonance (SPR) sensor and biotinylated inositol phosphate. The equilibrium dissociation constants obtained using this approach reflected the distinct magnitude of acyl group-based and phosphate group-based interactions. The dissociation constant (K-D) for Pr55(Gag) complexed with 1,4,5-IP3 (an inositol with divalent phosphate groups and devoid of lipid groups) was 2170 mu M, while the K-D for di-C-8-P1 (a lipid-containing inositol devoid of divalent phosphate groups) was 186 mu M, and the K-D for di-C-8-P1(4,5)P2 (an inositol with both lipid and divalent phosphate groups) was 47.4 mu M. The same trend in affinity was observed when these phosphoinositides were complexed with MA. Our results suggest that the contribution of hydrophobic acyl chains is greater than negatively charged inositol phosphates in Pr550(Gag)/MA binding. Furthermore, each inositol phosphate (devoid of lipid groups) tested showed a distinct Pr55(Gag)-binding affinity depending on the position and number of phosphate groups. However, the position and number of phosphate groups had no effect on MA-binding affinity.