Bourhis Jean-Marie, Johansson Kenth, Receveur-Bréchot Véronique, Oldfield Christopher J, Dunker Keith A, Canard Bruno, Longhi Sonia
Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, Cedex 09 13288, Marseille, France.
Virus Res. 2004 Feb;99(2):157-67. doi: 10.1016/j.virusres.2003.11.007.
The nucleoprotein of measles virus consists of an N-terminal domain, N(CORE) (aa 1-400), resistant to proteolysis, and a C-terminal domain, N(TAIL) (aa 401-525), hypersensitive to proteolysis and not visible by electron microscopy. Using two complementary computational approaches, we predict that N(TAIL) belongs to the class of natively unfolded proteins. Using different biochemical and biophysical approaches, we show that N(TAIL) is indeed unstructured in solution. In particular, the spectroscopic and hydrodynamic properties of N(TAIL) indicate that this protein domain belongs to the premolten globule subfamily within the class of intrinsically disordered proteins. The isolated N(TAIL) domain was shown to be able to bind to its physiological partner, the phosphoprotein (P), and to undergo an induced folding upon binding to the C-terminal moiety of P [J. Biol. Chem. 278 (2003) 18638]. Using a computational analysis, we have identified within N(TAIL) a putative alpha-helical molecular recognition element (alpha-MoRE, aa 488-499), which could be involved in binding to P via induced folding. We report the bacterial expression and purification of a truncated form of N(TAIL) (N(TAIL2), aa 401-488) devoid of the alpha-MoRE. We show that N(TAIL2) has lost the ability to bind to P, thus supporting the hypothesis that the alpha-MoRE may play a role in binding to P. We have further analyzed the alpha-helical propensities of N(TAIL2) and N(TAIL) using circular dichroism in the presence of 2,2,2-trifluoroethanol. We show that N(TAIL2) has a lower alpha-helical potential compared to N(TAIL), thus suggesting that the alpha-MoRE may be indeed involved in the induced folding of N(TAIL).
麻疹病毒的核蛋白由一个对蛋白水解有抗性的N端结构域N(CORE)（氨基酸1 - 400）和一个对蛋白水解高度敏感且在电子显微镜下不可见的C端结构域N(TAIL)（氨基酸401 - 525）组成。使用两种互补的计算方法，我们预测N(TAIL)属于天然未折叠蛋白类别。通过不同的生化和生物物理方法，我们表明N(TAIL)在溶液中确实是无结构的。特别是，N(TAIL)的光谱和流体动力学性质表明该蛋白结构域属于内在无序蛋白类别中的前熔球亚家族。分离出的N(TAIL)结构域被证明能够与其生理伴侣磷蛋白（P）结合，并在与P的C端部分结合时发生诱导折叠[《生物化学杂志》278 (2003) 18638]。通过计算分析，我们在N(TAIL)中鉴定出一个假定的α - 螺旋分子识别元件（α - MoRE，氨基酸488 - 499），它可能通过诱导折叠参与与P的结合。我们报告了一种缺失α - MoRE的N(TAIL)截短形式（N(TAIL2)，氨基酸401 - 488）的细菌表达和纯化。我们表明N(TAIL2)失去了与P结合的能力，从而支持了α - MoRE可能在与P结合中起作用的假设。我们在2,2,2 - 三氟乙醇存在的情况下使用圆二色性进一步分析了N(TAIL2)和N(TAIL)的α - 螺旋倾向。我们表明与N(TAIL)相比，N(TAIL2)具有较低的α - 螺旋潜力，因此表明α - MoRE可能确实参与了N(TAIL)的诱导折叠。
