Investigation of the binding geometry of a peripheral membrane protein
Investigation of the binding geometry of a peripheral membrane protein
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DOI：
10.1021/bi051127
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发表时间：
2005-12-13
期刊：
影响因子：
2.9
通讯作者：
Kutateladze, TG
中科院分区：
文献类型：
--
作者：
Brunecky, R;Lee, S;Kutateladze, TG
越来越多的模块，包括FYVE结构域通过特异性识别脂质头基和疏水性插入双层将关键信号蛋白靶向膜。尽管膜插入在这些模块的功能中起着关键作用，但膜对接和渗透的结构机制仍不清楚。特别是，插入的蛋白质相对于膜表面的三维取向难以定量定义。在这里，我们确定了早期内体抗原1（EEAI）FYVE结构域的胶束渗透的几何形状，通过获得NMR衍生的限制，与蛋白质骨架酰胺和自旋标记探针之间的距离。将5-和14-doxyl-磷脂酰胆碱自旋标记物掺入到十二烷基磷酸胆碱（DPC）胶束中，并测量由于顺磁弛豫增强引起的FYVE结构域的酰胺信号强度的降低。相对于分子轴的FYVE结构域插入的载体估计，通过最小化的顺磁性限制中获得的磷脂酰肌醇3-磷酸（Pl 3 P）富集胶束仅含有DPC或与磷脂酰丝氨酸（PS）混合。额外的距离限制，获得使用一种新的自旋标记模拟PI（3）P，含有一个硝酰基自由基附近的苏糖醇组的脂质。胶束相互作用后，检测到指示膜插入环（MIL）的伸长的构象变化，其中环的疏水残基倾向于移动到胶束的非极性核心更深处。本研究中定义的FYVE结构域的胶束插入机制与突变数据和化学位移扰动一致，并证明了使用自旋标记核磁共振方法研究外周膜蛋白结合几何形状的优势。
A growing number of modules including FYVE domains target key signaling proteins to membranes through specific recognition of lipid headgroups and hydrophobic insertion into bilayers. Despite the critical role of membrane insertion in the function of these modules, the structural mechanism of membrane docking and penetration remains unclear. In particular, the three-dimensional orientation of the inserted proteins with respect to the membrane surface is difficult to define quantitatively. Here, we determined the geometry of the micelle penetration of the early endosome antigen 1 (EEAI) FYVE domain by obtaining NMR-derived restraints that correlate with the distances between protein backbone amides and spin-labeled probes. The 5- and 14-doxyl-phosphatidylcholine spin-labels were incorporated into dodecylphosphocholine (DPC) micelles, and the reduction of amide signal intensities of the FYVE domain due to paramagnetic relaxation enhancement was measured. The vector of the FYVE domain insertion was estimated relative to the molecular axis by minimizing the paramagnetic restraints obtained in phosphatidylinositol 3-phosphate (Pl3P)-enriched micelles containing only DPC or mixed with phosphatidylserine (PS). Additional distance restraints were obtained using a novel spin-label mimetic of PI(3)P that contains a nitroxyl radical near the threitol group of the lipid. Conformational changes indicative of elongation of the membrane insertion loop (MIL) were detected upon micelle interaction, in which the hydrophobic residues of the loop tend to move deeper into the nonpolar core of micelles. The micelle insertion mechanism of the FYVE domain defined in this study is consistent with mutagenesis data and chemical shift perturbations and demonstrates the advantage of using the spin-label NMR approach for investigating the binding geometry by peripheral membrane proteins.