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首页> 外文期刊>Journal of Molecular Biology >Structure and dynamics of micelle-bound neuropeptide Y: Comparison withunligated NPY and implications for receptor selection
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Structure and dynamics of micelle-bound neuropeptide Y: Comparison withunligated NPY and implications for receptor selection

机译：胶束结合神经肽Y的结构和动力学：与未连接的NPY的比较及其对受体选择的影响

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摘要

The biological importance of the neuropeptide Y (NPY) has steered a number of investigations about its solution structure over the last 20 years. Here, we focus on the comparison of the structure and dynamics of NPY free in solution to when bound to a membrane mimetic, dodecyl-phosphocholine (DPC) micelles, as studied by 2D H-1 NMR spectroscopy. Both, free in solution and in the micelle-bound form, the N-terminal seg ment (Tyr1-Glu15) is shown to extend like a flexible tail in solution. This is not compatible with the PP-fold model for NPY that postulates back-folding of the flexible N terminus onto the C-terminal helix. The correlation time (tau (c)) of NPY in aqueous solution, 5.5 (+/-1.0) ns at 32 degreesC, is only consistent with its existence in a dimeric form. Exchange contributions especially enhancing transverse relaxation rates (R-2) of residues located on one side of the C-terminal helix of the molecule are supposed to originate from dimerization of the NPY molecule. The dimerization interface was directly probed by looking at N-15-labeled NPY/spin-labeled [TOAC34]-[N-14]-NPY heterodimers and revealed both parallel and antiparallel alignment of the helices. The NMR-derived three-dimensional structure of micelle-bound NPY at 37 degreesC and pH 6.0 is similar but not identical to that free in solution. The final set of 17 lowest-energy DYANA structures is particularly well defined in the region of residues 21-31, with a mean pairwise RMSD of 0.23 Angstrom for the backbone heavy atoms and 0.85 Angstrom for all heavy atoms. The combination of NMR relaxation data and CD measurements clearly demonstrates that the a-helical region Ala18-Thr32 is more stable, and the C-terminal tetrapeptide becomes structured only in the presence of the phosphocholine micelles. The position of NPY relative to the DPC micelle surface was probed by adding micelle integrating spin labels. Together with information from H-1,H-2 exchange rates, we conclude that the interaction of NPY with the micelle is promoted by the amphiphilic alpha -helical segment of residues Tyr21-Thr32. NPY is located at the lipid-water interface with its C-terminal helix parallel to the membrane surface and penetrates the hydrophobic interior only via insertions of a few long aliphatic or aromatic side-chains. From these data we can demonstrate that the dimer interface of neuropeptide Y is similar to the interface of the monomer binding to DPC-micelles. We speculate that binding of the NPY monomer to the membrane is an essential kev step preceeding receptor binding, thereby pre-orientating the C-terminal tetrapeptide and possibly inducing the bio-active conformation.
机译：在过去的20年中，神经肽Y（NPY）的生物学重要性引起了许多有关其溶液结构的研究。在这里，我们专注于溶液中游离NPY的结构和动力学的比较，该过程与通过2D H-1 NMR光谱学研究的结合到膜模拟物十二烷基磷酸胆碱（DPC）胶束上时的比较。 N末端片段（Tyr1-Glu15）在溶液中呈游离态并以胶束结合形式存在，都像溶液中的柔性尾巴一样延伸。这与NPY的PP折叠模型不兼容，后者假定柔性N末端向后折叠到C末端螺旋上。 NPY在水溶液中的相关时间（tau（c））在32摄氏度时为5.5（+/- 1.0）ns，仅与其以二聚体形式存在的时间一致。交换贡献，特别是提高位于分子C末端螺旋一侧的残基的横向弛豫速率（R-2），被认为源自NPY分子的二聚化。通过查看N-15标记的NPY /自旋标记的[TOAC34]-[N-14] -NPY异二聚体直接探测二聚化界面，并揭示了螺旋的平行和反平行排列。在37℃和pH 6.0下，胶束结合的NPY的NMR三维结构与溶液中游离的NMR相似但不相同。最后一组17个最低能量的DYANA结构在残基21-31的区域中定义得特别好，主链重原子的平均成对RMSD为0.23埃，所有重原子的平均成对RMSD为0.85埃。 NMR弛豫数据和CD测量值的结合清楚地表明，α-螺旋区Ala18-Thr32更稳定，并且C端四肽仅在存在磷酸胆碱胶束的情况下才结构化。 NPY相对于DPC胶束表面的位置通过添加整合自旋标记的胶束来探测。连同来自H-1，H-2汇率的信息，我们得出结论，NPY与胶束的相互作用是由残基Tyr21-Thr32的两亲性α-螺旋片段促进的。 NPY位于脂质-水界面，其C末端螺旋平行于膜表面，仅通过插入一些长的脂族或芳族侧链即可渗透疏水性内部。从这些数据中，我们可以证明神经肽Y的二聚体界面类似于结合DPC胶束的单体界面。我们推测，NPY单体与膜的结合是在受体结合之前必不可少的步骤，从而使C末端四肽预先取向，并可能诱导生物活性构象。

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