The elusive middle domain of Hsp104 and ClpB: location and function - PubMed (original) (raw)

Review

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

Morgan E Desantis et al. Biochim Biophys Acta. 2012 Jan.

Abstract

Hsp104 in yeast and ClpB in bacteria are homologous, hexameric AAA+ proteins and Hsp100 chaperones, which 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.

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Figures

Figure 1. In vitro disaggregase activity of Hsp104 and ClpB

(a) Hsp104 remodels amorphous aggregates via collaboration with Hsp70 and Hsp40, while ClpB remodels these types of aggregates via collaboration with DnaK, DnaJ, and GrpE (KJE). The product of disaggregation of amorphous aggregates is natively folded protein. Hsp104 or ClpB hexamer is shown in gray with the front half cutaway to reveal the axial channel running down the length of the structure. Four pore loops with conserved tyrosines residues are shown in orange. These pore loops are important for substrate binding and threading through the axial channel. (b) Only Hsp104 is able to remodel amyloid aggregates and in vitro this can proceed without the aid of Hsp70 and Hsp40. Products of amyloid disaggregation are soluble natively folded protein and fragmented amyloids. However, for some amyloids (e.g. Sup35 prions) remodeling can continue to generate disordered-type aggregates that Hsp104 cannot remodel and which lack the seeding activity of amyloid [23].

Figure 2. Predicted structures and hexameric models of Hsp104 and ClpB

(a) Domain organization of one monomer of Hsp104 and ClpB. N-terminal domain (N) shown in purple, Nucleotide binding domain 1 (NBD1) shown in cyan, Middle domain (MD) in yellow, Nucleotide binding domain 2 (NBD2) in dark blue. Only Hsp104 has the short C-terminal extension (C) shown in green. Sequence numbering for ClpB is shown on top and for Hsp104 is shown on the bottom. (b) TClpB crystal structure. Domain coloring corresponds with part (a). A 180° rotation about the vertical axis is shown on the right. (c) The Tsai model for the hexameric quaternary structure of TClpB. The Tsai model, which was initially based on Cryo-EM envelopes generated with TClpB is shown on left. (d) The Saibil model, which used Hsp104 to generate Cryo-EM density, is shown in the middle. (e) The 6.93Å crystal structure of hexameric, full length ClpC is shown on the right. The adaptor protein MecA was omitted for clarity. A side view is shown on top and a view down the axial channel from the N-terminus is shown on the bottom. One subunit is colored as described in part (a). The other five subunits are in gray.

Figure 3. Middle domain nomenclature

(a) An alignment of the MD from E. coli ClpB, T. thermophilus ClpB, and S. cerevisiae Hsp104. Helix 1 is colored in green, helix 2 is colored in purple, helix 3 is colored in light blue, and helix four is colored in yellow. Motif 1 (also called wing 2) is boxed in black while motif 2 (also called wing 1) is boxed in orange. Arrowheads denote key residues discussed in the text. (b) Close up of the MD in the TClpB crystal structure. Each helix and motif is colored as indicated in part (a). NBD2 is omitted for clarity. Arrows point to side chains (shown as sticks) of key residues discussed in the text.

Figure 4. Overlay of ClpB and ClpC monomers

ClpB and ClpC monomers were aligned by their AAA+ domains using Pymol. The resulting RMSD was 3.4. Domains for ClpB are colored as in Figure 2 with the N domain in purple, NBD1 in cyan, MD in yellow, and NBD2 in dark blue. All domains in ClpC are colored in orange. Note the drastically different position of the ClpB N-terminal domain (marked with an arrow) and the different angle of the ClpC MD (marked with an asterisk).

Figure 5. Summary of ClpB and Hsp104 chimera activity

Cartoon representation of the domain chimeras utilized in references [51, 68, 69]. Green cylinders represent Hsp104 domains and blue cylinders represent ClpB domains. Activity relative to wild-type (WT) Hsp104 is shown in green symbols and activity relative to WT ClpB is shown in blue symbols. ++ indicates that the chimera displayed greater activity than WT, + indicates that the chimera displayed comparable activity to WT, − indicates that chimera displayed less activity than WT, while Ø indicates that no activity was observed for the chimera tested. Blank cells indicate that chimera was not tested for this activity. [PSI+] propagation, [RNQ+] propagation, ATPase activity, increase in ATPase activity upon binding α-casein, the ability to bind α-casein, conferred thermotolerance in E. coli, conferred thermotolerance in S. cerevisiae, and association with DnaK as measured by a bacterial 2-hybrid system were tested. Additionally, DnaK, DnaJ, and GrpE and Hsp70/40 mediated disaggregation of Green fluorescent protein (GFP), β-galactosidase (β-Gal), and malate dehydrogenase (MDH) were tested.

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The elusive middle domain of Hsp104 and ClpB: location and function - PubMed (original) (raw)