From the Department of Structural Biology and Pittsburgh Center for HIV-Host Protein Interactions, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261.
J Biol Chem. 2014 Jan 31;289(5):2577-88. doi: 10.1074/jbc.M113.534453. Epub 2013 Dec 8.
The human homolog of the yeast DNA repair protein RAD23, hHR23A, has been found previously to interact with the human immunodeficiency virus, type 1 accessory protein Vpr. hHR23A is a modular protein containing an N-terminal ubiquitin-like (UBL) domain and two ubiquitin-associated domains (UBA1 and UBA2) separated by a xeroderma pigmentosum complementation group C binding (XPCB) domain. All domains are connected by flexible linkers. hHR23A binds ubiquitinated proteins and acts as a shuttling factor to the proteasome. Here, we show that hHR23A utilizes both the UBA2 and XPCB domains to form a stable complex with Vpr, linking Vpr directly to cellular DNA repair pathways and their probable exploitation by the virus. Detailed structural mapping of the Vpr contacts on hHR23A, by NMR, revealed substantial contact surfaces on the UBA2 and XPCB domains. In addition, Vpr binding disrupts an intramolecular UBL-UBA2 interaction. We also show that Lys-48-linked di-ubiquitin, when binding to UBA1, does not release the bound Vpr from the hHR23A-Vpr complex. Instead, a ternary hHR23A·Vpr·di-Ub(K48) complex is formed, indicating that Vpr does not necessarily abolish hHR23A-mediated shuttling to the proteasome.
先前发现酵母 DNA 修复蛋白 RAD23 的人类同源物 hHR23A 与人类免疫缺陷病毒 1 型辅助蛋白 Vpr 相互作用。hHR23A 是一种模块化蛋白，包含一个 N 端泛素样（UBL）结构域和两个泛素相关结构域（UBA1 和 UBA2），由一个着色性干皮病互补组 C 结合（XPCB）结构域隔开。所有结构域都由柔性接头连接。hHR23A 结合泛素化蛋白，作为穿梭因子到蛋白酶体。在这里，我们表明 hHR23A 利用 UBA2 和 XPCB 结构域与 Vpr 形成稳定的复合物，将 Vpr 直接连接到细胞 DNA 修复途径及其可能被病毒利用。通过 NMR 对 hHR23A 上 Vpr 接触点进行详细的结构映射，揭示了 UBA2 和 XPCB 结构域上的大量接触表面。此外，Vpr 结合破坏了 UBL-UBA2 结构域之间的分子内相互作用。我们还表明，当结合到 UBA1 时，K48 连接的二泛素不会将结合的 Vpr 从 hHR23A-Vpr 复合物中释放出来。相反，形成了一个三元 hHR23A·Vpr·二泛素（K48）复合物，表明 Vpr 不一定会废除 hHR23A 介导的向蛋白酶体的穿梭。
