ATP modulates holdase activity of fungal and human 110-kilodalton heat shock proteins to promote protein folding.

Heat shock proteins of the 70-kDa family (Hsp70s) are highly abundant and conserved molecular chaperones that help preserve proteostasis primarily by facilitating proper protein folding. Heat shock protein of the 110-kDa family (Hsp110s), a specialized branch of the Hsp70/Hsp110 superfamily, function both as nucleotide exchange factor (NEF) cochaperones for Hsp70s and as independent "holdase" chaperones that stabilize non-native polypeptides to prevent aggregation and facilitate downstream refolding by Hsp70s. While Hsp110 NEF activity is well characterized, the consequences of adenosine 5'-triphosphate (ATP) binding for Hsp110 holdase behavior have remained largely unexplored. Although holdase activity is generally considered nucleotide-independent, reports of ATP-dependent effects have raised questions about the underlying mechanism. Here, we examined the biochemical properties of Multicopy Suppressor of ira1 3 (Msi3), the sole Hsp110 in Candida albicans, to dissect the role of ATP in holdase function. We first identified an inhibitory effect of elevated Mg2+ concentrations on Msi3 holdase activity. This inhibitory effect is counteracted by the intrinsically disordered C-terminal segment, revealing a distinct stabilization role for this region, previously of unknown function. In addition, ATP alleviates inhibition by elevated Mg2+, providing an explanation for an apparent ATP-dependence observed previously. Interestingly, although dispensable for aggregation suppression, ATP modulates Msi3 holdase activity for refolding competence by broadening the concentration range over which it remains productive. Increasing Msi3 concentration improved overall downstream refolding recovery but slowed refolding kinetics, and ATP alleviated this kinetic constraint. Analyses of Hsp105, the major human Hsp110, suggest that these biochemical properties are largely conserved. Together, these findings suggest that ATP modulates Hsp110 holdase activity by tuning the balance between substrate sequestration and engagement dynamics, revealing an ATP-dependent regulatory dimension of Hsp110 holdase function that is mechanistically distinct from its NEF activity.