Institut de Biologie Structurale, Université Grenoble 1, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France.
J Mol Biol. 2012 Jul 20;420(4-5):310-23. doi: 10.1016/j.jmb.2012.04.023. Epub 2012 Apr 26.
Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here, we report on the results of an investigation of an intrinsically disordered 188-residue fragment of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline Src homology 3 (SH3) binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic-resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1 (bridging integrator protein 1), a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a noncanonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.
理解病毒复制和粒子组装所涉及的分子机制具有首要的基础和医学重要性。固有构象无序在病毒蛋白及其与其他病毒和宿主细胞蛋白的相互作用中起着重要作用，通过瞬态存在的结构元件。在这里，我们报告了对丙型肝炎病毒非结构蛋白 5A（NS5A）的 188 个残基的无规卷曲片段的研究结果，该片段包含一个经典的多脯氨酸Src 同源 3（SH3）结合基序，使用了灵敏度和分辨率优化的多维 NMR 方法，并结合了小角度 X 射线散射数据。我们的研究提供了关于瞬态局部和长程结构以及该 NS5A 片段的快速时间尺度动力学的详细原子分辨率信息。此外，我们可以描述与 Bin1（桥连整合蛋白 1）的 SH3 结构域的两种不同的相互作用模式，Bin1 是一种促凋亡的肿瘤抑制因子。尽管该蛋白大部分是无序的，但它包含三个区域，这些区域会暂时采用α-螺旋结构，部分由远程三级相互作用稳定。这两个瞬态α-螺旋形成了一个非典型的 SH3 结合基序，允许低亲和力的 SH3 结合。我们的研究结果有助于更好地理解 NS5A 蛋白在丙型肝炎病毒感染中的作用。目前的工作还突出了 NMR 光谱学的强大功能，它可以用于描述多个结合事件，包括球状和高度无序蛋白之间的短寿命瞬态相互作用。
