Brunecky Roman, Lee Stephanie, Rzepecki Piotr W, Overduin Michael, Prestwich Glenn D, Kutateladze Andrei G, Kutateladze Tatiana G
Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA.
Biochemistry. 2005 Dec 13;44(49):16064-71. doi: 10.1021/bi051127+.
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 (EEA1) 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 (PI3P)-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.
越来越多包含FYVE结构域的模块通过对脂质头部基团的特异性识别以及疏水插入双层膜，将关键信号蛋白靶向到膜上。尽管膜插入在这些模块的功能中起着关键作用，但膜对接和穿透的结构机制仍不清楚。特别是，插入蛋白相对于膜表面的三维取向难以进行定量定义。在这里，我们通过获得与蛋白质主链酰胺和自旋标记探针之间距离相关的核磁共振衍生约束，确定了早期内体抗原1（EEA1）FYVE结构域的胶束穿透几何形状。将5-和14-二氧磷酰胆碱自旋标记物掺入十二烷基磷酰胆碱（DPC）胶束中，并测量由于顺磁弛豫增强导致的FYVE结构域酰胺信号强度的降低。通过最小化在仅含有DPC或与磷脂酰丝氨酸（PS）混合的富含磷脂酰肌醇3-磷酸（PI3P）的胶束中获得的顺磁约束，估计FYVE结构域插入相对于分子轴的向量。使用一种新型的PI(3)P自旋标记模拟物获得了额外的距离约束，该模拟物在脂质的苏糖醇基团附近含有一个硝酰自由基。在胶束相互作用时检测到了表明膜插入环（MIL）伸长的构象变化，其中环的疏水残基倾向于更深地移动到胶束的非极性核心中。本研究中定义的FYVE结构域的胶束插入机制与诱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。
