Regulatory Circuits of the AAA+ Disaggregase Hsp104

Abstract

Yeast Hsp104 is an AAA؉ chaperone that rescues proteins from the aggregated state. Six protomers associate to form the functional hexamer. Each protomer contains two AAA؉ modules, NBD1 and NBD2. Hsp104 converts energy provided by ATP into mechanical force used to thread polypeptides through its axial channel, thereby disrupting protein aggregates. But how the action of its 12 AAA؉ domains is co-ordinated to catalyze disaggregation remained unexplained. Here, we identify a sophisticated allosteric network consisting of three distinct pathways that senses the nucleotide state of AAA؉ modules and transmits this information across the Hsp104 hexamer. As a result of this communication, NBD1 and NBD2 each adopt two distinct conformations (relaxed and tense) that are reciprocally regulated. The key element in the network is the NBD1-ATP state that enables Hsp104 to switch from a barely active [ AAAϩ proteins (ATPases associated with various cellular activities) comprise a functionally diverse family of molecular machines that share a large degree of sequence homology and an evolutionary conserved mechanism (1). AAAϩ proteins use chemical energy provided by ATP hydrolysis to catalyze forceinduced structural rearrangements of client molecules. However, the details of this mechano-chemical coupling are not well understood. AAAϩ proteins consist of one or two conserved ATP-binding modules (NBDs) 2 linked to function-specific auxiliary domains. Each AAAϩ module harbors canonical Walker A and Walker B motifs that are critical for nucleotide binding and hydrolysis, respectively. In many cases, AAAϩ proteins assemble into ring-shaped oligomers (2).Hsp104 from yeast and its bacterial homolog, ClpB, are AAAϩ chaperones that provide thermotolerance to their hosts by disassembling protein aggregates after heat shock (3-5). Reactivation of these proteins seems to be crucial because the removal of protein aggregates by degradation was found to be insufficient for restoring cell viability (6, 7). To disrupt noncovalent interactions between aggregated polypeptides, Hsp104 and ClpB are believed to employ a common mechanism that involves co-operation with the Hsp70 chaperone system (8, 9): ATP binding and hydrolysis induce a sequence of domain movements in Hsp104 which in turn exert a mechanical force on bound polypeptide substrates (10 -12). As a result, individual polypeptide chains are extracted from aggregates and threaded through the central channel of the disaggregase (6, 7, 13). This proposed mode of action is reminiscent of models established for other AAAϩ proteins, such as the unfoldase unit of the proteasome or the bacterial unfoldases ClpA and ClpX, which unfold target polypeptides and feed them into the proteolytic chamber of an associated protease (14 -17). Nucleotide-induced domain rearrangements have been observed for several AAAϩ proteins (18,19), but to what extent these movements occur in a co-ordinated fashion and how AAAϩ machines couple chemical energy to mechanical work are largely unknown. For ClpX it has been su...

Allosteric Communication between the Nucleotide Binding Domains of Caseinolytic Peptidase B

“…The fact that ADP binding to NBD2 does not modify the affinity of NBD1 for this nucleotide points out that the ATP-and ADP-induced allosteric signal transmitted from NBD2 to NBD1 is different, and therefore, it could help to establish the asymmetric functioning of both NBDs in the hexamer. Functional asymmetry has been recently proposed for Hsp104, as the TϽ-ϾR transition at both NBDs seems to be inversely linked (25). Data presented here also indicate that ATP binding to NBD2 induces an allosteric signal that promotes cooperative binding of ATP and substrates to the NBD1 ring.…”

“…ATP hydrolysis at NBD2 and, therefore, exchange of ATP by ADP increases the k cat of NBD1 activating its hydrolysis. A recent study has identified cis and trans protomer interactions that regulate the activity of Hsp104 (25). Intrasubunit communication has also been proposed for ClpB Th as the ATPase activity of NBD2 is stimulated after an M-domain conformational change triggered by ATP binding to NBD1 (26).…”

Structural basis for intersubunit signaling in a protein disaggregating machine

“…We propose that this signaling network is crucial to sense the nucleotide state in the adjacent subunit and to reset the nucleotide cycle in the ClpB ring following ATP hydrolysis. The existence of an ISS network that regulates ATP hydrolysis in diverse AAA+ ring complexes is also consistent with a sequential ATP-hydrolysis mechanism proposed for ClpB (45,46) and Hsp104 (47), with four out of six subunits in the ClpB homohexamer occupied by nucleotides at any one time (46). This model is similar to the staircase mechanism proposed for the T7 gene 4 ring helicase (48), and is consistent with the nucleotide occupancy observed in the crystal structure of an engineered, covalently linked ClpX hexamer (11).…”

Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation

“…Because a knockout of Hsp101 was not successful, we generated dominant-negative variants of the protein (Walker A point mutation; see SI Materials and Methods, Generation of Dominant-Negative Walker A Point Mutations, for details). Formation of heterooligomers composed of the wild-type protein and the dominant-negative variant has been shown to inactivate the disaggregase (35,61). When we expressed dominant-negative Hsp101 in a Q103-GFP background, foci formation during heat stress was not altered.…”