Solution structure of mouse HBS1L/SKI7-specific UBA domain in complex with ubiquitin: Implications for stalled ribosome recognition.

Abstract

Human HBS1L and SKI7 (HBS1LV3) are isoforms encoded by the same gene. HBS1L forms a complex with PELO to recognize ribosomes stalled on non-stop mRNAs and promotes ribosome splitting, whereas SKI7 acts as a bridge between the exosome and the SKI complex to mediate mRNA decay on stalled ribosomes. Despite substantial differences in the sequence and function of their C-terminal regions, the two isoforms share an identical N-terminal domain (termed UBAh) that resembles the ubiquitin binding UBA and CUE domains (collectively referred to as the three-helix bundle ubiquitin-binding [THB-Ub] group). Although UBAh has been predicted to interact with ubiquitin moieties attached to the small subunits of stalled ribosomes, evidence for its interaction with ubiquitin is lacking. Herein, we report the NMR structure of the mouse UBAh-ubiquitin complex. UBAh adopts a three-helix bundle architecture (α1-α2-α3) with unique connecting loops. The hydrophobic patch in UBAh interacts with the Ile44-centered hydrophobic patch of ubiquitin in a binding mode nearly identical to that of the UBA and CUE domains. In contrast, the α1/α2 loop contains a distinctive double β-turn that accommodates the protrusion of the ubiquitin β-turn. The hallmark motif of UBAh, located within and downstream of this loop, was identified as VLGD/E. HSQC titration experiments yielded a dissociation constant of approximately 50 µM for ubiquitin. These findings demonstrate that UBAh specifically interacts with ubiquitin in vitro, providing structural insights into its potential role in recruiting HBS1L-PELO and SKI7 to stalled ribosomes.