Sólyom Zsófia, Ma Peixiang, Schwarten Melanie, Bosco Michaël, Polidori Ange, Durand Grégory, Willbold Dieter, Brutscher Bernhard
Institut de Biologie Structurale, Université Grenoble 1, Grenoble, France; Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France; Centre National de Recherche Scientifique, Grenoble, France.
Institut de Biologie Structurale, Université Grenoble 1, Grenoble, France; Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France; Centre National de Recherche Scientifique, Grenoble, France; Institute of Complex Systems-6 Structural Biochemistry, Forschungszentrum Jülich, Jülich, Germany.
Biophys J. 2015 Oct 6;109(7):1483-96. doi: 10.1016/j.bpj.2015.06.040.
Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein's interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.
内在无序蛋白（IDP）在不具备明确三维结构的情况下履行其生理功能。尽管如此，IDP的残余结构和构象动力学对于其功能背后的机制至关重要。例如，瞬时二级结构区域通常参与分子识别，其结构在结合时会稳定（或不稳定）。另一方面，长程相互作用决定了IDP的流体力学半径，从而决定了蛋白质通过所谓的抛锚机制捕获结合伙伴的距离。长程相互作用的调节还提供了一种微调蛋白质相互作用网络的便捷方式，通过使结合位点或多或少易于接近。在这里，我们主要通过核磁共振光谱研究了丙型肝炎病毒（HCV）非结构蛋白5A（NS5A）中的残余二级结构和长程相互作用，NS5A是一种典型的病毒IDP，在病毒生命周期中具有多种功能。NS5A包括一个N端折叠结构域，随后是一个大的（约250个残基）无序C端部分。将全长NS5A的核磁共振光谱与仅由C端残基191 - 447组成的蛋白质构建体（NS5A - D2D3）的光谱进行比较，使我们得出结论，NS5A的球状部分和无序部分之间没有显著相互作用。NS5A - D2D3尽管整体具有高度灵活性，但显示出很大程度的局部残余（α螺旋和β转角）结构以及静电长程相互作用网络。此外，我们可以证明，这些长程相互作用在与宿主蛋白Bin1结合后以及NS5A被CK2磷酸化后会发生调节。由于参与这些相互作用的带电肽区域在不同的HCV基因型中高度保守，这些瞬时长程相互作用可能对NS5A在HCV生命周期中的某些功能很重要。
