Institute of Human Virology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Bioorg Med Chem. 2013 Jun 15;21(12):3443-9. doi: 10.1016/j.bmc.2013.04.035. Epub 2013 Apr 24.
The dengue capsid protein C is a highly basic alpha-helical protein of ~100 amino acid residues that forms an emphipathic homodimer to encapsidate the viral genome and to interact with viral membranes. The solution structure of dengue 2 capsid protein C (DEN2C) has been determined by NMR spectroscopy, revealing a large dimer interface formed almost exclusively by hydrophobic residues. The only acidic residue (Glu87) conserved in the capsid proteins of all four serotypes of dengue virus forms a salt bridge with the side chains of Lys45 and Arg55'. To understand the structural and functional significance of this conserved salt bridge, we chemically synthesized an N-terminally truncated form of DEN2C ((WT)DEN2C) and its salt bridge-void analog (E87A)DEN2C using the native chemical ligation technique developed by Kent and colleagues. Comparative biochemical and biophysical studies of these two synthetic proteins using circular dichroism spectroscopy, fluorescence polarization, protein thermal denaturation, and proteolytic susceptibility assay demonstrated that the conserved salt bridge contributed to DEN2C dimerization and stability as well as its resistance to proteolytic degradation. Our work provided insight into the role of a fully conserved structural element of the dengue capsid protein C and paved the way for additional functional studies of this important viral protein.
登革热外壳蛋白 C 是一种高度碱性的α-螺旋蛋白，约有 100 个氨基酸残基，形成一个同型二聚体来包裹病毒基因组，并与病毒膜相互作用。登革热 2 型外壳蛋白 C（DEN2C）的溶液结构已通过 NMR 光谱学确定，揭示了一个主要由疏水性残基形成的大二聚体界面。在所有四种血清型登革热病毒的外壳蛋白中保守的唯一酸性残基（Glu87）与 Lys45 和 Arg55'的侧链形成盐桥。为了了解这个保守盐桥的结构和功能意义，我们使用 Kent 及其同事开发的天然化学连接技术，通过化学合成方法合成了 DEN2C 的 N 端截断形式（WT）DEN2C 和其盐桥缺失类似物（E87A）DEN2C。使用圆二色性光谱、荧光偏振、蛋白质热变性和蛋白水解敏感性测定对这两种合成蛋白进行比较生化和生物物理研究表明，保守盐桥有助于 DEN2C 的二聚化和稳定性，以及其对蛋白水解降解的抗性。我们的工作深入了解了登革热外壳蛋白 C 中完全保守结构元件的作用，并为进一步研究这个重要的病毒蛋白的功能铺平了道路。
