Gralle Matthias, Oliveira Cristiano L P, Guerreiro Luiz H, McKinstry William J, Galatis Denise, Masters Colin L, Cappai Roberto, Parker Michael W, Ramos Carlos H I, Torriani Iris, Ferreira Sérgio T
Instituto de Bioquímica Médica, Programa de Bioquímica e Biofísica Celular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21944-590, Brazil.
J Mol Biol. 2006 Mar 24;357(2):493-508. doi: 10.1016/j.jmb.2005.12.053. Epub 2006 Jan 3.
Proteolytic cleavage of the amyloid precursor protein (APP) by beta and gamma-secretases gives rise to the beta-amyloid peptide, considered to be a causal factor in Alzheimer's disease. Conversely, the soluble extracellular domain of APP (sAPPalpha), released upon its cleavage by alpha-secretase, plays a number of important physiological functions. Several APP fragments have been structurally characterized at atomic resolution, but the structures of intact APP and of full-length sAPPalpha have not been determined. Here, ab initio reconstruction of molecular models from high-resolution solution X-ray scattering (SAXS) data for the two main isoforms of sAPPalpha (sAPPalpha(695) and sAPPalpha(770)) provided models of sufficiently high resolution to identify distinct structural domains of APP. The fragments for which structures are known at atomic resolution were fitted within the solution models of full-length sAPPalpha, allowing localization of important functional sites (i.e. glycosylation, protease inhibitory and heparin-binding sites). Furthermore, combined results from SAXS, analytical ultracentrifugation (AUC) and size-exclusion chromatography (SEC) analysis indicate that both sAPPalpha isoforms are monomeric in solution. On the other hand, SEC, bis-ANS fluorescence, AUC and SAXS measurements showed that sAPPalpha forms a 2:1 complex with heparin. A conformational model for the sAPPalpha:heparin complex was also derived from the SAXS data. Possible implications of such complex formation for the physiological dimerization of APP and biological signaling are discussed in terms of the structural models proposed.
β-分泌酶和γ-分泌酶对淀粉样前体蛋白（APP）进行蛋白水解切割会产生β-淀粉样肽，该肽被认为是阿尔茨海默病的致病因素。相反，APP经α-分泌酶切割后释放的可溶性细胞外结构域（sAPPα）具有多种重要的生理功能。多个APP片段已在原子分辨率下进行了结构表征，但完整APP和全长sAPPα的结构尚未确定。在这里，通过对sAPPα的两种主要异构体（sAPPα(695)和sAPPα(770)）的高分辨率溶液X射线散射（SAXS）数据进行分子模型的从头重建，得到了分辨率足够高的模型，以识别APP的不同结构域。将已知原子分辨率结构的片段拟合到全长sAPPα的溶液模型中，从而确定重要功能位点（即糖基化、蛋白酶抑制和肝素结合位点）的位置。此外，SAXS、分析超速离心（AUC）和尺寸排阻色谱（SEC）分析的综合结果表明，两种sAPPα异构体在溶液中均为单体。另一方面，SEC、双-ANS荧光、AUC和SAXS测量表明，sAPPα与肝素形成2:1复合物。还从SAXS数据推导出了sAPPα:肝素复合物的构象模型。根据所提出的结构模型，讨论了这种复合物形成对APP生理二聚化和生物信号传导的可能影响。
