The elusive middle domain of Hsp104 and ClpB: Location and function

Abstract

Hsp104 in yeast and ClpB in bacteria are homologous, hexameric AAA+ proteins and Hsp100 chaperones that function in the stress response as ring-translocases that drive protein disaggregation and reactivation. Both Hsp104 and ClpB contain a distinctive coiled-coil middle domain (MD) inserted in the first AAA+ domain, which distinguishes them from other AAA+ proteins and Hsp100 family members. Here, we focus on recent developments concerning the location and function of the MD in these hexameric molecular machines, which remains an outstanding question. While the atomic structure of the hexameric assembly of Hsp104 and ClpB remains uncertain, recent advances have illuminated that the MD is critical for the intrinsic disaggregase activity of the hexamer and mediates key functional interactions with the Hsp70 chaperone system (Hsp70 and Hsp40) that empower protein disaggregation.

Engineering enhanced protein disaggregases for neurodegenerative disease

“…67,82,[151][152][153][154] Importantly, the MD is less conserved than the 2 NBDs, indicating that it can withstand various missense mutations without eliminating disaggregase functionality. 67 Indeed, the MD can even tolerate large protein insertions (e.g., insertion of lysozyme between Asn467 and Glu468 in MD helix 2) or helix replacements and yet still maintain Hsp104 disaggregase activity. 106,152,153 Moreover, previous studies suggested that Hsp104 MD variants can have unexpected gain-of-function phenotypes, including rescue of polyglutamine aggregation and toxicity.…”

“…67,81 We focused our libraries on the coiled-coil MD, which is comprised of 4 a-helices and facilitates optimal ATPase activity, communication between NBD1 and NBD2, intrinsic disaggregase activity, and interactions with Hsp70 during disordered aggregate dissolution. 67,82,[151][152][153][154] Importantly, the MD is less conserved than the 2 NBDs, indicating that it can withstand various missense mutations without eliminating disaggregase functionality. 67 Indeed, the MD can even tolerate large protein insertions (e.g., insertion of lysozyme between Asn467 and Glu468 in MD helix 2) or helix replacements and yet still maintain Hsp104 disaggregase activity.…”

“…65,66 Hsp104 forms a ring-shaped complex that disaggregates protein by coupling ATP hydrolysis to partial or complete substrate translocation across its central channel. 21,[67][68][69][70][71][72] Optimal Hsp104 activity is typically achieved in conjunction with the Hsp110, Hsp70, and Hsp40 chaperone system. 65,[73][74][75][76] Importantly, Hsp104 is the only cellular agent known to rapidly dissolve stable amyloid fibrils, 73,77,88 which can self-template their own cross-b conformation and encode transmissible phenotypes.…”

Reversing deleterious protein aggregation with re-engineered protein disaggregases

“…30 The protein forms a 2-tiered hexamer with a central pore, through which substrate is threaded. 30,31 Hsp104 is very large, consisting of 6 102-kDa monomers, and its structure remains poorly understood. Therefore, Hsp104 is a relatively poor candidate for rational design.…”

“…We selected the middle domain of Hsp104 for randomization, because the middle domain facilitates ATPase activity, communication between NBD1 and NBD2, intrinsic disaggregase activity, and interactions with Hsp70. 26,30 Restricting mutations to just the middle domain was key, as limiting the size of the randomized region allowed for greater depth of coverage. Additionally, this strategy allowed for sequencing to be restricted to a length allowing a single sequencing reaction per variant.…”