Breuer Sebastian, Gerlach Holger, Kolaric Branko, Urbanke Claus, Opitz Norbert, Geyer Matthias
Abteilungen Physikalische Biochemie und Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Biochemistry. 2006 Feb 21;45(7):2339-49. doi: 10.1021/bi052052c.
The accessory HIV-1 Nef protein is essential for viral replication, high virus load, and progression to AIDS. These functions are mediated by the alteration of signaling and trafficking pathways and require the membrane association of Nef by its N-terminal myristoylation. However, a large portion of Nef is also found in the cytosol, in line with the observation that myristoylation is only a weak lipidation anchor for membrane attachment. We performed biochemical studies to analyze the implications of myristoylation on the conformation of Nef in aqueous solution. To establish an in vivo myristoylation assay, we first optimized the codon usage of Nef for Escherichia coli expression, which resulted in a 15-fold higher protein yield. Myristoylation was achieved by coexpression with the N-myristoyltransferase and confirmed by mass spectrometry. The myristoylated protein was soluble, and proton NMR spectra confirmed proper folding. Size exclusion chromatography revealed that myristoylated Nef appeared of smaller size than the unmodified form but not as small as an N-terminally truncated from of Nef that omits the anchor domain. Western blot stainings and limited proteolysis of both forms showed different recognition profiles and degradation pattern. Analytical ultracentrifugation revealed that myristoylated Nef prevails in a monomeric state while the unmodified form exists in an oligomeric equilibrium of monomer, dimer, and trimer associations. Finally, fluorescence correlation spectroscopy using multiphoton excitation revealed a shorter diffusion time for the lipidated protein compared to the unmodified form. Taken together, our data indicated myristoylation-dependent conformational changes in Nef, suggesting a rather compact and monomeric form for the lipidated protein in solution.
HIV-1辅助蛋白Nef对病毒复制、高病毒载量以及发展为艾滋病至关重要。这些功能是通过信号传导和运输途径的改变介导的，并且需要Nef通过其N端肉豆蔻酰化作用与膜结合。然而，很大一部分Nef也存在于细胞质中，这与肉豆蔻酰化只是一种较弱的膜附着脂质化锚定的观察结果一致。我们进行了生化研究，以分析肉豆蔻酰化对Nef在水溶液中构象的影响。为了建立体内肉豆蔻酰化测定法，我们首先优化了Nef在大肠杆菌表达中的密码子使用，这使蛋白质产量提高了15倍。通过与N-肉豆蔻酰转移酶共表达实现肉豆蔻酰化，并通过质谱法确认。肉豆蔻酰化的蛋白质是可溶的，质子核磁共振光谱证实其折叠正确。尺寸排阻色谱显示，肉豆蔻酰化的Nef比未修饰形式的尺寸小，但不像省略锚定结构域的Nef N端截短形式那么小。两种形式的蛋白质印迹染色和有限蛋白酶解显示出不同的识别谱和降解模式。分析超速离心显示，肉豆蔻酰化的Nef以单体状态为主，而未修饰形式则存在于单体、二聚体和三聚体缔合的寡聚平衡中。最后，使用多光子激发的荧光相关光谱显示，与未修饰形式相比，脂化蛋白质的扩散时间更短。综上所述，我们的数据表明Nef中存在肉豆蔻酰化依赖性构象变化，表明溶液中脂化蛋白质呈相当紧凑的单体形式。
