The M-domain controls Hsp104 protein remodeling activity in an Hsp70/Hsp40-dependent manner

Abstract

Yeast Hsp104 is a ring-forming ATP-dependent protein disaggregase that, together with the cognate Hsp70 chaperone system, has the remarkable ability to rescue stress-damaged proteins from a previously aggregated state. Both upstream and downstream functions for the Hsp70 system have been reported, but it remains unclear how Hsp70/Hsp40 is coupled to Hsp104 protein remodeling activity. Hsp104 is a multidomain protein that possesses an N-terminal domain, an M-domain, and two tandem AAA(+) domains. The M-domain forms an 85-A long coiled coil and is a hallmark of the Hsp104 chaperone family. While the three-dimensional structure of Hsp104 has been determined, the function of the M-domain is unclear. Here, we demonstrate that the M-domain is essential for protein disaggregation, but dispensable for Hsp104 ATPase- and substrate-translocating activities. Remarkably, replacing the Hsp104 M-domain with that of bacterial ClpB, and vice versa, switches species specificity so that our chimeras now cooperate with the noncognate Hsp70/DnaK chaperone system. Our results demonstrate that the M-domain controls Hsp104 protein remodeling activities in an Hsp70/Hsp40-dependent manner, which is required to unleash Hsp104 protein disaggregating activity.

Fig. 1

Disaggregation of model substrates by Hsp104-T4L fusion proteins. (a-c) Reactivation of protein aggregates by Hsp104, Hsp104_T4L, ΔN-Hsp104, or ΔN-Hsp104_T4L (1 μM each) alone and together with Hsp70 and Ydj-1 (1 μM each). Assays contained an ATP-regenerating system (20 mM phosphoenolpyruvate and 1 μM pyruvate kinase) and 4 mM ATP. Proteins were produced as described in the supplementary material. (a) Reactivation of heat-aggregated β-gal was determined as described and is shown over time as percentage of a native control. Standard errors of three independent assays are shown. (b) 4.5 μM enhanced GFP (BioVision, Mountain View, CA) was denatured at 80 °C in buffer A (25 mM HEPES pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)₂, and 10 mM DTT) for 10 min and diluted 1:10 with buffer A containing chaperones as indicated. The increase in fluorescence was measured with a Synergy HT microplate reader (BioTek, Winooski, VT) in 1 min intervals. A representative data set of three experiments is shown. (c) 2 μM MDH (Roche Diagnostics, Indianapolis, IN) was incubated in buffer A at 47 °C for 30 min and diluted 2.5-fold into buffer A at 22 °C with chaperones as indicated. MDH activity was determined after 180 min as described and is shown as percentage of a native control. Standard errors of three independent assays are shown.

Fig. 2

Biochemical characterization of Hsp104 variants. (a) Domain arrangements of Hsp104, Hsp104ΔM, and Hsp104-ClpB chimeras. The AAA-1 domain is colored blue, deletion of the M-domain is indicated by a grey line, and domains that were replaced by corresponding ClpB sequences are colored orange. Hsp104ΔM and Hsp104-ClpB chimeras were constructed by cassette mutagenesis, expressed, and purified as described in the supplementary material. (b) Hsp104, Hsp104ΔM, and Hsp104-ClpB chimeras (0.6 mg/ml) were incubated for 20 min at 22 °C in buffer A containing 2 mM ATPγS and 0.2 mg/ml κ-casein. Hexamer assembly was analyzed by size-exclusion chromatography on a Superdex 200 10/300 GL column (GE Healthcare, Piscataway, NJ) in buffer A plus 10 μM ATPγS. (c) ATPase rates of Hsp104, Hsp104ΔM, and Hsp104-ClpB chimeras in the absence or presence of 0.2 mg/ml κ-casein were determined using a coupled ATP-regenerating system at 30 °C in buffer A containing 2 mM ATP as described. Standard errors of three independent assays are shown. (d) Reactivation of aggregated model substrates by Hsp70 and Ydj-1 (1 μM each) alone and together with either Hsp104 (1 μM) or Hsp104ΔM (1 μM). Disaggregation assays were performed as described in Fig. 1a-c. Standard errors of three independent assays are shown. (e) Fluorescence polarization binding isotherms of FITC-casein to Hsp104_Trap, Hsp104ΔM_Trap, and Hsp104BM_Trap. Equilibrium binding constants were determined at 22 °C by adding 0.15 µg/ml FITC-casein (Sigma-Aldrich, St. Louis, MO) to Trap hexamers that were preassembled with 2 mM ATP in buffer A at 22 °C. Standard errors of three independent assays are shown.

Fig. 3

M-domain replacement switches the species-specificity of the bi-chaperone system. (a) Reactivation of chemically denatured FFL over time by Hsp104 or Hsp104-ClpB chimeras (1 μM each) alone and together with Hsp70 and Ydj-1 (1 μM each) or DnaK (0.86 μM), DnaJ (0.43 μM), and GrpE (0.25 μM). DnaK, DnaJ, and GrpE were purified as described in the supplementary material. 10 μM FFL (Promega, Madison, WI) was denatured in 7 M Urea, 25 mM HEPES pH 7.5, 50 mM KCl, 15 mM MgCl₂, 10 mM DTT, and 5 mM ATP at 22 °C, and diluted 125-fold into 25 mM HEPES pH 7.5, 150 mM KCl, 15 mM MgCl₂, 2 mM DTT and chaperones as indicated. Luminescence was measured at 22 °C as described. (b) Reactivation of heat-aggregated MDH by Hsp104 or Hsp104-ClpB chimeras (1 μM each) alone and together with Hsp70 and Ydj-1 (1 μM each) or DnaK (0.86 μM), DnaJ (0.43 μM), and GrpE (0.25 μM). Assay conditions were the same as in Fig. 1c. (c) Reactivation of chemically denatured FFL over time by ClpB or ClpBHM (1 μM each) alone and together with DnaK (0.86 μM), DnaJ (0.43 μM), and GrpE (0.25 μM) or Hsp70 and Ydj-1 (1 μM each). ClpB and ClpBHM were purified as described in the supplementary material. Assay conditions were the same as in Fig. 3a. (d) Reactivation of heat-aggregated MDH by ClpB or ClpBHM (1 μM each) alone and together with DnaK (0.86 μM), DnaJ (0.43 μM), and GrpE (0.25 μM) or Hsp70 and Ydj-1 (1 μM each). Assay conditions were the same as in Fig. 1c. Standard errors of three independent assays are shown in Fig. 3a-d.

Fig. 4

Disaggregation requires M-domain mediated collaboration with the Hsp70/DnaK system. (a) Degradation of FITC-casein (0.12 mg/ml) by ClpP (2 μM) alone and together with HAP_Trap, HAP, HAPΔM, or HAP-ClpB chimeras (1 μM each). FITC-casein was incubated in buffer A at 22 °C for 60 min with chaperones as indicated, together with an ATP-regenerating system (6 mM phosphoenolpyruvate and 0.5 μM pyruvate kinase), and 3 mM ATP. Reactions were analyzed as described by measuring the fluorescence signal of acid-soluble peptides using a LS55 fluorescence spectrometer (Perkin Elmer, Waltham, MA). Standard errors of three independent assays are shown. (b) Degradation of heat-aggregated FAM-labeled MDH (1.5 μM; labeling is described in the supplementary material) by ClpP (2 μM) and HAP variants (1 μM) alone and together with DnaK (0.86 μM), DnaJ (0.43 μM), and GrpE (0.25 μM) or Hsp70 and Ydj-1 (1 μM each). Reactions were set up and analyzed the same way as described for the FITC-casein degradation assay (Fig. 4a). Standard errors of three independent assays are shown.