Functional analysis of conserved cis- and trans-elements in the Hsp104 protein disaggregating machine

Abstract

Hsp104 is a double ring-forming AAA+ ATPase, which harnesses the energy of ATP binding and hydrolysis to rescue proteins from a previously aggregated state. Like other AAA+ machines, Hsp104 features conserved cis- and trans-acting elements, which are hallmarks of AAA+ members and are essential to Hsp104 function. Despite these similarities, it was recently proposed that Hsp104 is an atypical AAA+ ATPase, which markedly differs in 3D structure from other AAA+ machines. Consequently, it was proposed that arginines found in the non-conserved M-domain, but not the predicted Arg-fingers, serve the role of the critical trans-acting element in Hsp104. While the structural discrepancy has been resolved, the role of the Arg-finger residues in Hsp104 remains controversial. Here, we exploited the ability of Hsp104 variants featuring mutations in one ring to retain ATPase and chaperone activities, to elucidate the functional role of the predicted Arg-finger residues. We found that the evolutionarily conserved Arg-fingers are absolutely essential for ATP hydrolysis but are dispensable for hexamer assembly in Hsp104. On the other hand, M-domain arginines are not strictly required for ATP hydrolysis and affect the ATPase and chaperone activities in a complex manner. Our results confirm that Hsp104 is not an atypical AAA+ ATPase, and uses conserved structural elements common to diverse AAA+ machines to drive the mechanical unfolding of aggregated proteins.

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

“…His 6 -tagged Hsp70, Hsp104, and their variants were bacterially overexpressed as described (50). Proteins were purified from cleared lysates by Ni-nitrilotriacetic acid (NTA) affinity and anion exchange chromatography.…”

“…For enzymatic assays, Hsp104 and mutants were used after the second Ni-NTA column. His 6 -Hsp40 (S. cerevisiae Ydj1) and EGFP, which contained a TEV cleavable N-terminal His 7 -tag and the first 15 amino acids of RepA at the C terminus (EGFP), were purified essentially as described (6,50).…”

“…Protein Disaggregation Assay. Protein disaggregation assays with chemically denatured FFL or heat-aggregated β-gal were performed as described (50). α-Glucosidase aggregates were prepared by denaturing 0.5 μM yeast α-glucosidase (Sigma-Aldrich) at 50°C for 12 min in assay buffer.…”

“…α-Glucosidase aggregates were prepared by denaturing 0.5 μM yeast α-glucosidase (Sigma-Aldrich) at 50°C for 12 min in assay buffer. Heataggregated α-glucosidase (0.2 μM) was added to assay buffer containing equimolar concentrations of molecular chaperones (0.7 μM monomer) and a previously described ATP regenerating system (50). Recovered α-glucosidase activity was assayed by adding 1 mM 4-nitrophenyl-α-N-glucopyranoside, incubating for 15 min at 30°C, and measuring the absorbance at 405 nm.…”

“…Heat-aggregated EGFP (0.225 μM) was added to 0.5 μM (monomer) of molecular chaperones in the presence of an ATP regenerating system. Real-time recovery of EGFP fluorescence was monitored at 25°C over 120 min as described (50).…”