Global unfolding of a substrate protein by the Hsp100 chaperone ClpA

Abstract

The bacterial protein ClpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP (refs 1, 2). ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences^3,4,5, much as the 19S ATPase ‘cap’ of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins^6,7,8. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins^9,10; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates¹¹. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder^12,13,14. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation^5,15. Fluorescence studies both with and without a ‘trap’ version of the chaperonin GroEL, which binds non-native forms of GFP¹⁶, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.