Abstract
<jats:title>ABSTRACT</jats:title> <jats:p>AAA+ proteases maintain proteostasis by mechanically unfolding protein substrates before degradation, yet how ATP-driven pulling is converted into productive unfolding remains poorly understood. Here, using ssrA-tagged GFP substrates spanning a range of surface charges, we show that electrostatic complementarity between the positively charged ClpX substrate-entry channel and folded substrate domains governs unfolding efficiency. Positively charged substrates unfolded inefficiently despite substrate recognition, thermal stability, ATPase activation, and pore-loop engagement comparable to negatively charged substrates. Cryo-EM structures of a positively charged substrate revealed multiple substrate-engagement states arising from electrostatic incompatibility at the ClpX substrate-entry channel, whereas negatively charged substrates formed favorable electrostatic interactions that likely stabilize partially unfolded intermediates during successive ATP-driven unfolding attempts. Together, these findings identify electrostatic complementarity as a key determinant of productive ATP-driven protein unfolding and suggest that tuning substrate or substrate-entry electrostatics provides a mechanism for regulating AAA+ protease activity.</jats:p>