AAA unfoldases and disaggregases

HSP100 disaggregases - Nature's most powerful cleaning machines

HSP100 unfoldases employ a powerful mechanism to recover proteins from aggregates. Upon forming a hexameric ring, the AAA proteins unravel polypeptides by threading them through a narrow central pore. Substrate stretching and unfolding is mediated by ATP-driven power strokes, which result from the movement of rigid ATPase bodies composed of the large (L) subdomain of one protomer and the small (S) subdomain of the next. Coordination of adjacent L/S* modules (the asterisk denotes the neighboring subunit) relies on a special active site organization, as each nucleotide binding site is formed by residues of the L-, S*- and L*-subdomains at the subunit interface (Fig.XXX).

In terms of substrates, the particularly powerful HSP100 unfoldases have the remarkable ability to disentangle protein aggregates, which, owing to their inert, scrambled, and water-insoluble character, represent the most challenging target for the protein quality control system. Although HSP100 chaperones play a crucial function in removing these potentially dangerous aggregates, the molecular details of their robust cleaning activity have remained unclear. It has been postulated that the disaggregation activity of HSP100 machines relies on the presence of two AAA rings, most likely to provide a strong, 2-handed grip for remodeling protein substrates. To avoid damage to native proteins, the high unfolding potential of Hsp104 and related disaggregases needs to be carefully regulated. This control is mediated by an inserted coiled-coil domain (M-domain, MD), which assembles a molecular belt encircling the hexameric particle. Binding of the Hsp70 chaperone to the MD activates Hsp104 and targets it towards protein aggregates.

 

Our findings: 2016 Mechanisms underlying and controlling HSP100 disaggregase activity

 

 

2016 Structural organization of the Hsp104 disaggregase

To investigate how protein aggregates are enzymatically dissolved, we performed a structure-function analysis of the fungal Hsp104, a disaggregase that was originally identified as a critical factor for survival under extreme stress conditions and, later, for prion propagation. To this end, we analyzed the crystal structure of Hsp104 from Chaetomium thermophilum that – although forming a helical filament – reveals important mechanistic features. First, we identified the long-sought mechanical link coupling the two AAA rings of HSP100 chaperones and, second, we delineated structural details underlying the regulatory role of the MD. Jointly, these two elements make Hsp104 a very potent yet highly tunable protein disaggregase.

The PS1 hairpin (shown with electron density) protrudes from AAA2 to AAA1 coordinating the activities of the two ATPase rings.
Hsp104 self-entrapment: The coiled-coil domain assemble a ring around the Hsp104 hexamer that sterically controls the mobility of the engaged ATPase bodies.

1) Synchronizing ATPase engines in complex molecular machines: The Hsp104 crystal structure identified the structural motif that mechanically links the two ATPase engines of a HSP100 chaperone machine. We show that a characteristic beta-hairpin, referred to as pre-sensor-1 (PS1) motif, couples conformational changes in AAA1 with the repositioning of the catalytic sensor-1 residue of AAA2. The signaling mechanism relies on the rearrangement of individual beta-strands within the AAA2 beta-sheet. We hypothesized that the relatively short parallel beta-sheet found in the AAA fold is well suited for such a distortion. Notably, the PS1-hairpin is the defining feature of a major AAA superclade comprising also single-ring ATPase machines of various remodeling functions. We thus proposed that the PS1-hairpin and the connected sensor-1 residue constitute a common signaling device to couple ATPase activity with external stimuli such as substrate, co-factor, or ligand binding. It is this multi-purpose structural device that provides the long-sought link between the two AAA1 and AAA2 rings in the powerful protein unfolding machine Hsp104.

2) Self-entrapment by the MD: Hsp104 and its orthologs are under control of their MD extension that can form a continuous ring around the hexamer. Binding of Hsp70 to the MD opens this ring and stimulates disaggregase activity by an unknown mechanism. Our data demonstrated that the MDs compose a topological belt tightly embracing the AAA1 ring. Each MD glues two ATPase L/S* modules together, thus restricting their relative movement and keeping the AAA machine in a latent state. Hsp70 binding would lead to a rearrangement of the MD motif disrupting its contacts with AAA1. The now released ATPase modules are free to fulfill their dynamic task in the protein unfolding and disaggregation reaction. As it is assumed that chaperones, unfoldases, and proteases acting on damaged proteins must be highly flexible to be active, it will be interesting to see whether other quality control factors are regulated by similar steric-restraint mechanisms.

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