Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity

Abstract

Two members of the AAA+ superfamily, ClpB and Hsp104, collaborate with Hsp70 and Hsp40 to rescue aggregated proteins. However, the mechanisms that elicit and underlie their proteinremodeling activities remain unclear. We report that for both Hsp104 and ClpB, mixtures of ATP and ATPγS unexpectedly unleash activation, disaggregation, and unfolding activities independent of co-chaperones. Mutations reveal how remodeling activities are elicited by impaired hydrolysis at individual nucleotide binding domains. However, for some substrates, mixtures of ATP and ATPγS abolish remodeling, while for others ATP binding without hydrolysis is sufficient. Remodeling of different substrates necessitates a diverse balance of polypeptide holding (which requires ATP binding but not hydrolysis) and unfolding (which requires ATP hydrolysis). We suggest that this versatility in reaction mechanism enables ClpB and Hsp104 to reactivate the entire aggregated proteome after stress, and enables Hsp104 to control prion inheritance.Life demands that members of the AAA+ ATPase superfamily (ATPases associated with various cellular activities) couple energy from ATP hydrolysis to the remodeling of a bewildering array of macromolecular structures, that range from protein to DNA and RNA 1, 2 . Typically, eukaryotic genomes encode 50-80 family members 1 , each of which occupies specific niches that require specialized modes of substrate selection and regulation. The extraordinary adaptive radiation of AAA+ proteins to function in a multitude of cellular reactions illustrates the versatility of their structurally conserved AAA+ domain. Subunits containing AAA+ domains assemble into oligomeric rings, and ATP binds at the interface between adjacent protomers 1, 2 . AAA+ oligomers undergo considerable conformational changes during ATP binding and hydrolysis, although how these events are regulated and transduced into productive substrate remodeling remains largely enigmatic. Furthermore, it remains unanswered whether individual AAA+ family members rely on a common reaction mechanism to remodel various macromolecular clients. It is also unclear whether different AAA+ members have evolved distinct methods to engage and restructure substrates, or if individual proteins can switch between distinct reaction mechanisms for different substrates.Two members of the AAA+ superfamily separated by ~2 billion years of evolution 3 , yeast Hsp104, and its E. coli homolog, ClpB, allow cell survival after exposure to extreme environmental stress 4-7 . They function to dissolve and renature thousands of diverse 5 Correspondence: Sue Wickner,

Hsp104 Targets Multiple Intermediates on the Amyloid Pathway and Suppresses the Seeding Capacity of Aβ Fibrils and Protofibrils

“…However, Hsp104 is thought to catalyze substrate unfolding by actively processing polypeptides through the central pore of its hexameric form with a substrate binding state that requires ATP or ATPγS. 4,5,7,36 Thus, the nucleotideindependent effect observed in the current work suggests a different type of substrate interaction of Hsp104 that results in an efficient inhibition of amyloid fibril formation.…”

“…2,3 Hsp104 and its prokaryotic homologue ClpB are hexameric AAA + ATPases with two nucleotide-binding domains (NBDs) per subunit that couple asymmetric cycles of ATP hydrolysis to the disaggregation of their polypeptide substrates. The substrate binding of ClpB/Hsp104 was shown to be dependent on the ATP state (Scheme 1a); accordingly, reduced or impaired ATP hydrolysis, as in the case of the ATPase mutant E285Q/E687Q (Hsp104 TRAP ), increases the affinity towards polypeptide substrates 5,7,8 (Scheme 1b). The processing of polypeptides by wild-type ClpB/Hsp104 results in the unfolding of non-native structures and separation of single polypeptides from aggregates, thus facilitating the refolding of proteins.…”

Collaboration between the ClpB AAA+ remodeling protein and the DnaK chaperone system

“…ClpB and Hsp104 have recently been shown to possess protein remodeling activities in vitro in the absence of the DnaK/Hsp70 system, including protein activation, protein unfolding and reactivation of heat-inactivated proteins (22). These activities are elicited under conditions that asymmetrically slow ATP hydrolysis at some but not all of the 12 ATP binding sites of the hexameric holoenzyme (22).…”

“…This condition very likely allows polypeptides to be held, a process that requires ATP binding but not hydrolysis, and unfolded, a reaction that requires energy derived from ATP hydrolysis. The protein remodeling activities of ClpB and Hsp104 are also expressed when one of the two nucleotide-binding domains is defective as the result of mutation (22).…”

“…These activities are elicited under conditions that asymmetrically slow ATP hydrolysis at some but not all of the 12 ATP binding sites of the hexameric holoenzyme (22). One condition that induces the protein remodeling activities of ClpB and Hsp104 is the use of mixtures of ATP and ATP␥S, a slowly hydrolyzable ATP analog, as the nucleotide source (22). This condition very likely allows polypeptides to be held, a process that requires ATP binding but not hydrolysis, and unfolded, a reaction that requires energy derived from ATP hydrolysis.…”