The N-terminal domain of the endoplasmic reticulum-resident chaperone Grp94 functions in aggregation prevention.

Glucose-regulated protein 94 (Grp94), an Hsp90 molecular chaperone localized in the endoplasmic reticulum, plays a central role in maintaining cellular proteostasis by facilitating protein folding and refolding in a nucleotide-dependent manner. Beyond its folding functions, Grp94 also prevents the aggregation of client proteins under stress conditions; however, the mechanistic basis of this aggregation-prevention activity is not fully understood. In this study, we investigated the requirements of Grp94's prevention of aggregation activity and identified the functional domain responsible using in vitro light scattering and functional assays with two model client proteins, citrate synthase and luciferase. The results show that Grp94 suppresses the thermal aggregation of clients independently of ATP-binding, ATP-hydrolysis and calcium binding. Notably, the aggregation prevention activity for both clients is localized to the N-terminal domain of Grp94, where a peptide-binding deficient mutant (H146D) shows enhanced aggregation suppression at limiting chaperone concentrations. This enhanced prevention of aggregation activity is attributed to increased affinity for misfolded clients, which is likely due to decreased client dissociation rates. Consequently, client release by the Grp94 peptide-binding mutant is inhibited, thereby impairing downstream remodeling by the BiP chaperone system. While the N-terminal domain of Grp94 appears to be a general requirement in the prevention of aggregation, the involvement of Grp94's pre-N domain is likely client specific. Collectively, these findings provide new insights into the aggregation-prevention function of Grp94 and implicate a previously identified peptide-binding site in aggregation prevention activity.