GRP1 pleckstrin homology domain: Activation parameters and novel search mechanism for rare target lipid
GRP1 pleckstrin homology domain: Activation parameters and novel search mechanism for rare target lipid
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DOI：
10.1021/bi049017a
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发表时间：
2004-12-28
期刊：
影响因子：
2.9
通讯作者：
Falke, JJ
中科院分区：
文献类型：
--
作者：
Corbin, JA;Dirkx, RA;Falke, JJ
普列克底物蛋白同源性（PH）结构域通过结合磷脂酰肌醇磷酸（PIP）脂质家族的第二信使脂质在广泛的信号传导途径中发挥中心作用。给定类型的PIP脂质在特定的细胞膜中形成，其中它通常是主体脂质混合物的次要组分。例如，信号传导脂质PI（3，4，5）P-3（或PIP 3）主要在质膜的内小叶中产生，据信其在质膜的内小叶中从未超过本体脂质的0.02%。本研究的重点是磷酸肌醇，亚型1（GRP 1），调节肌动蛋白细胞骨架在质膜表面的PIP 3信号的一般受体的PH域。本研究系统地分析了GRP 1-PH结构域与PIP脂质靶点在双层表面结合的平衡和动力学特征。利用蛋白质-膜荧光共振能量转移（FRET）检测GRP 1-PH结构域与膜结合PIP脂质的对接的平衡结合测量证实了与PIP 3的特异性结合。一种新的FRET竞争性结合测量，开发定量对接亲和力产生的K-D为50 +/- 10 nM的GRP 1-PH结构域结合到膜结合的PIP 3在生理脂质混合物接近质膜内小叶的组成。该观察到的KD位于通过生理PIP 3信号调节的合适范围内。有趣的是，当从脂质混合物中除去背景阴离子脂质磷脂酰丝氨酸（PS）和磷脂酰肌醇（PI）时，相互作用的亲和力降低至少12倍。停流动力学研究，使用蛋白质-膜FRET监测协会和解离的时间进程表明，这种亲和力的降低产生于相应的减少GRP 1-PH结构域对接的关闭率从膜结合的PIP 3结构域解离的变化很小或没有。总的来说，这些发现表明PH结构域不仅与其靶脂质相互作用，而且与膜表面的其他特征相互作用。结果是一致的，与以前未描述的类型的两步搜索机制的脂质结合域，其中弱，非特异性的PH域和背景阴离子脂质之间的静电相互作用，促进搜索的膜表面的PIP 3头基，从而加快高亲和力，特异性对接的域，其罕见的目标脂质。
Pleckstrin homology (PH) domains play a central role in a wide array of signaling pathways by binding second messenger lipids of the phosphatidylinositol phosphate (PIP) lipid family. A given type of PIP lipid is formed in a specific cellular membrane where it is generally a minor component of the bulk lipid mixture. For example, the signaling lipid PI(3,4,5)P-3 (or PIP3) is generated primarily in the inner leaflet of the plasma membrane where it is believed to never exceed 0.02% of the bulk lipid. The present study focuses on the PH domain of the general receptor for phosphoinositides, isoform 1 (GRP1), which regulates the actin cytoskeleton in response to PIP3 signals at the plasma membrane surface. The study systematically analyzes both the equilibrium and kinetic features of GRP1-PH domain binding to its PIP lipid target on a bilayer surface. Equilibrium binding measurements utilizing protein-to-membrane fluorescence resonance energy transfer (FRET) to detect GRP1-PH domain docking to membrane-bound PIP lipids confirm specific binding to PIP3. A novel FRET competitive binding measurement developed to quantitate docking affinity yields a K-D of 50 +/- 10 nM for GRP1-PH domain binding to membrane-bound PIP3 in a physiological lipid mixture approximating the composition of the plasma membrane inner leaflet. This observed KD lies in a suitable range for regulation by physiological PIP3 signals. Interestingly, the affinity of the interaction decreases at least 12-fold when the background anionic lipids phosphatidylserine (PS) and phosphatidylinositol (PI) are removed from the lipid mixture. Stopped-flow kinetic studies using protein-to-membrane FRET to monitor association and dissociation time courses reveal that this affinity decrease arises from a corresponding decrease in the on-rate for GRP1-PH domain docking with little or no change in the off-rate for domain dissociation from membrane-bound PIP3. Overall, these findings indicate that the PH domain interacts not only with its target lipid, but also with other features of the membrane surface. The results are consistent with a previously undescribed type of two-step search mechanism for lipid binding domains in which weak, nonspecific electrostatic interactions between the PH domain and background anionic lipids facilitate searching of the membrane surface for PIP3 headgroups, thereby speeding the high-affinity, specific docking of the domain to its rare target lipid.