<scp>ATP</scp> modulates holdase activity of fungal and human 110‐kilodalton heat shock proteins to promote protein folding

, 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.