The Vps27/Hrs/STAM (VHS) domain of the signaltransducing adaptor molecule (STAM) directs associated molecule with the SH3 domain of STAM (AMSH) specificity to longer ubiquitin chains and dictates the position of cleavage

The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation bySTAMproteins and the role of theSTAMVps27/ Hrs/STAM domain.Weshow that, in the presence ofSTAM,the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the k_cat of Lys⁶³-linked ubiquitin chain cleavage was comparable for di- and tri-ubiquitin, the K_m value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys⁶³-linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys⁶³-linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage.