Structural basis of HSP90C, a highly active chloroplastic HSP90 chaperone from Arabidopsis thaliana.

Chloroplasts are the main energy-producing organelles in plants, responsible for photosynthesis, CO2 fixation, and O2 production. These processes rely on the import of numerous nucleus-encoded proteins into the chloroplast and, eventually, the thylakoids. While translocation systems across chloroplast and thylakoid membranes are well characterized, the stromal route between these membranes remains poorly understood. The chloroplastic HSP90 (HSP90C) is likely to play a key role in this process, yet its structure and molecular mechanisms are unknown. Here, we combine structural and biophysical approaches to characterize HSP90C from Arabidopsis thaliana. We show that HSP90C displays exceptionally high ATPase activity compared with other HSP90 family members, driven by non-canonical mechanisms. These include an N-terminal disulfide bond that enhances ATPase activity and a C-terminal extension required for dimerization. These features arise from conserved sequence signatures shared among Angiospermae. Our work provides the first structural insights into HSP90C and advances understanding of chloroplast protein import mechanisms.