Structural plasticity of the membrane-bound protein degradation assembly supports bacterial adaptation to stress.

Protein degradation by ATPases associated with diverse cellular activities) (AAA+) proteases is essential for bacterial adaptation to stress. The membrane-bound protease FtsH forms an inner-membrane complex with the SPFH (stomatin, prohibitin, flotillin, and HflK/C) (SPFH) proteins HflK and HflC that promotes recovery from aminoglycoside antibiotics. Although open and closed HflK/C conformations have been described, their functional relevance has remained unclear. Here, we engineer a disulfide-crosslinked HflK/C variant to stabilize the closed state and determine its structure by high-resolution cryo-electron microscopy (cryo-EM). Cells expressing this variant, or an HflK/C mutant that disrupts FtsH binding, exhibit impaired growth under aminoglycoside stress, demonstrating that conformational dynamics and productive HflK/C-FtsH interactions are required for adaptation. Surprisingly, cryo-EM of the FtsH⋅HflK/C complex from tobramycin-treated cells reveals a distinct conformation with two openings that may facilitate substrate entry during proteotoxic stress. Together, these findings establish HflK/C conformational flexibility as a determinant of stress adaptation and provide a framework for understanding SPFH protein function.