Cryo-EM Structure of an Active Tetradecameric AAA+ Chaperone ClpL

Abstract

ATPases associated with diverse cellular activities (AAA+) chaperones are major component of Heat shock proteins (HSPs), participating in protein homeostasis. The functional oligomeric state of AAA+ chaperones is mostly modulated by their hexameric/dodecameric quaternary structures. However, little is known about non-hexameric/dodecameric AAA+ chaperones. Here we report the structural and biochemical characterizations of a tetradecameric AAA+ chaperone, ClpL from Streptococcus pneumoniae. ClpL plays an important role in escaping from host immune system when pathogenic pneumococci enter the host cell. We observed that ClpL exists as a tetradecamer in solution in the presence of ATP. The cryo-EM structure of ClpL reveals a striking tetradecameric arrangement. Solution structures of ClpL derived from small-angle X-ray scattering data suggest that the tetradecameric ClpL could assume a spiral conformation found in active hexameric/dodecameric AAA+ chaperone structures. Vertical positioning of the middle domain accounts for the head-to head arrangement of two heptameric rings. Biochemical activity assays with site-directed mutagenesis confirmed the critical roles of residues both in the integrity of the tetradecameric arrangement and activities of ClpL. Non-conserved Q321 and R670 are crucial in the heptameric ring assembly of ClpL. These results establish that ClpL is a functionally active tetradecamer, clearly distinct from hexameric/dodecameric AAA+ chaperones.