Université de Lyon, CNRS, UMR 5280 Institut des Sciences Analytiques, 69622 Villeurbanne, France.
J Biol Chem. 2012 May 25;287(22):18687-99. doi: 10.1074/jbc.M111.324954. Epub 2012 Apr 4.
As the upstream component of the ESCRT (endosomal sorting complexes required for transport) machinery, the ESCRT-0 complex is responsible for directing ubiquitinated membrane proteins to the multivesicular body pathway. ESCRT-0 is formed by two subunits known as Hrs (hepatocyte growth factor-regulated substrate) and STAM (signal transducing adaptor molecule), both of which harbor multiple ubiquitin-binding domains (UBDs). In particular, STAM2 possesses two UBDs, the VHS (Vps27/Hrs/Stam) and UIM (ubiquitin interacting motif) domains, connected by a 20-amino acid flexible linker. In the present study, we report the interactions of the UIM domain and VHS-UIM construct of STAM2 with monoubiquitin (Ub), Lys(48)- and Lys(63)-linked diubiquitins. Our results demonstrate that the UIM domain alone binds monoubiquitin, Lys(48)- and Lys(63)-linked diubiquitins with the same affinity and in the same binding mode. Interestingly, binding of VHS-UIM to Lys(63)-linked diubiquitin is not only avid, but also cooperative. We also show that the distal domain of Lys(63)-linked diubiquitin stabilizes the helical structure of the UIM domain and that the corresponding complex adopts a specific structural organization responsible for its greater affinity. In contrast, binding of VHS-UIM to Lys(48)-linked diubiquitin and monoubiquitin is not cooperative and does not show any avidity. These results may explain the better sorting efficiency of some cargoes polyubiquitinated with Lys(63)-linked chains over monoubiquitinated cargoes or those tagged with Lys(48)-linked chains.
作为 ESCRT（内体分选复合物必需的运输）机器的上游组件，ESCRT-0 复合物负责将泛素化的膜蛋白导向多泡体途径。ESCRT-0 由两个亚基组成，分别称为 Hrs（肝细胞生长因子调节的底物）和 STAM（信号转导衔接分子），它们都具有多个泛素结合结构域（UBD）。特别是，STAM2 具有两个 UBD，即 VHS（Vps27/Hrs/Stam）和 UIM（泛素相互作用模体）结构域，由一个 20 个氨基酸的柔性接头连接。在本研究中，我们报告了 STAM2 的 UIM 结构域和 VHS-UIM 结构与单泛素（Ub）、Lys（48）-和 Lys（63）-连接的二泛素的相互作用。我们的结果表明，UIM 结构域本身以相同的亲和力和相同的结合模式结合单泛素、Lys（48）-和 Lys（63）-连接的二泛素。有趣的是，VHS-UIM 与 Lys（63）-连接的二泛素的结合不仅是高亲和力的，而且是协同的。我们还表明，Lys（63）-连接的二泛素的远端结构域稳定了 UIM 结构域的螺旋结构，并且相应的复合物采用了特定的结构组织，这使其具有更高的亲和力。相比之下，VHS-UIM 与 Lys（48）-连接的二泛素和单泛素的结合不是协同的，也没有表现出任何高亲和力。这些结果可能解释了一些货物多聚泛素化的 Lys（63）-连接链比单泛素化货物或标记 Lys（48）-连接链具有更好的分拣效率的原因。
