Evidence for cooperative and domain-specific binding of the signal transducing adaptor molecule 2 (STAM2) to Lys⁶³-linked diubiquitin

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), Lys⁴⁸- and Lys⁶³-linked diubiquitins. Our results demonstrate that the UIM domain alone binds monoubiquitin, Lys⁴⁸- and Lys⁶³-linked diubiquitins with thesame affinity and in the same binding mode. Interestingly, binding of VHS-UIM to Lys⁶³-linked diubiquitin is not only avid, but also cooperative. We also show that the distal domain of Lys63-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 Lys⁴⁸-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 Lys⁶³-linked chains over monoubiquitinated cargoes or those tagged with Lys⁴⁸-linked chains.