Relevance of partially structured states in the non-classical secretion of acidic fibroblast growth factor.
Relevance of partially structured states in the non-classical secretion of acidic fibroblast growth factor.
复制标题
DOI：
10.1021/bi7002586
复制
发表时间：
2007-08
期刊：
影响因子：
2.9
通讯作者：
D. Rajalingam;I. Graziani;I. Prudovsky;C. Yu;T. Kumar
中科院分区：
文献类型：
--
作者：
D. Rajalingam;I. Graziani;I. Prudovsky;C. Yu;T. Kumar
酸性成纤维细胞生长因子(AFGF)是一种无信号肽的蛋白质，作为多蛋白释放复合体的一部分被分泌到细胞外间隙，由aFGF、S100A13(一种钙结合蛋白)和一种40 kDa(P40)形式的突触素(Syt1)组成，Syt1是一种参与各种分泌小泡对接的蛋白质。P40 Syt1，特别是其C2a结构域，被认为在aFGF和p40 Syt1与细胞膜内小叶磷脂相互作用所介导的aFGF释放复合体的非经典分泌中起主要作用。在本研究中，我们利用包括多维核磁共振光谱在内的多种生物物理技术，研究了酸性条件下aFGF和p40Syt1的C2a结构域的结构特征。尿素诱导的a成纤维细胞生长因子和C2a结构域的平衡展开(在pH 3.4时)都是非合作的，并继续积累稳定的中间态。1-苯胺基-8-萘磺酸盐(ANS)结合和尺寸排斥层析的结果表明，在酸性条件下(pH 3.4)，aFGF和C2a结构域都以部分结构状态存在。限制性胰酶消化分析和1H-15N化学位移扰动数据表明，在部分结构状态(S)下，蛋白质骨架的某些部分的灵活性增加。在碱性成纤维细胞生长因子中被部分结构状态(S)扰动的残基大多位于蛋白质的N-末端和C-末端。与之形成鲜明对比的是，稳定天然二级结构的大多数相互作用都保存在C2a结构域的部分结构化状态下。等温滴定量热数据表明，在pH为3.4时，aFGF与C2a结构域的结合亲和力显著增强。此外，a成纤维细胞生长因子和C2a结构域在部分结构状态下都表现出更高的脂结合亲和力。多蛋白的成纤维细胞生长因子释放复合体的跨膜移位似乎是由于a成纤维细胞生长因子和p40Syt1的C2a结构域的部分结构状态的形成。
Acidic fibroblast growth factor (aFGF) is a signal peptide-less protein that is secreted into the extracellular compartment as part of a multiprotein release complex, consisting of aFGF, S100A13 (a calcium binding protein), and a 40 kDa (p40) form of synaptotagmin (Syt1), a protein that participates in the docking of a variety of secretory vesicles. p40 Syt1, and specifically its C2A domain, is believed to play a major role in the non-classical secretion of the aFGF release complex mediated by the interaction of aFGF and p40 Syt1with the phospholipids of the cell membrane inner leaflet. In the present study, we investigate the structural characteristics of aFGF and the C2A domain of p40 Syt1 under acidic conditions, using a variety of biophysical techniques including multidimensional NMR spectroscopy. Urea-induced equilibrium unfolding (at pH 3.4) of both aFGF and the C2A domain are non-cooperative and proceed with the accumulation of stable intermediate states. 1-Anilino-8-napthalene sulfonate (ANS) binding and size-exclusion chromatography results suggest that both aFGF and the C2A domain exist as partially structured states under acidic conditions (pH 3.4). Limited trypsin digestion analysis and 1H-15N chemical shift perturbation data reveal that the flexibility of certain portions of the protein backbone is increased in the partially structured state(s) of aFGF. The residues that are perturbed in the partially structured state(s) in aFGF are mostly located at the N- and C-terminal ends of the protein. In marked contrast, most of the interactions stabilizing the native secondary structure are preserved in the partially structured state of the C2A domain. Isothermal titration calorimetry data indicate that the binding affinity between aFGF and the C2A domain is significantly enhanced at pH 3.4. In addition, both aFGF and the C2A domain exhibit much higher lipid binding affinity in their partially structured states. The translocation of the multiprotein FGF release complex across the membrane appears to be facilitated by the formation of partially structured states of aFGF and the C2A domain of p40 Syt1.