Electrostatic interactions between middle domain motif-1 and the AAA1 module of the bacterial ClpB chaperone are essential for protein disaggregation

Abstract

ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA⁺), ClpB forms a hexameric ring structure, with each protomer containing two AAA⁺ modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401-Arg-532 and the inter-subunit pair Asp-404-Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401-Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180-Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.

Keywords: ATPases associated with diverse cellular activities (AAA); Hsp104; chaperone; disaggregation; mass spectrometry (MS); protein aggregation; protein conformation; protein cross-linking; protein dynamic; structure-function.

Figure 1.

Positions of the mutated residues of TClpB. a, structure of TClpB (PDB code 1QVR) is shown. The N-terminal domain, AAA1, middle domain, and AAA2 are colored in green, blue, yellow, and red, respectively. b, structure of the TClpB middle domain. The amino acid residues, Glu-401, Glu-402, Asp-404, Glu-407, Arg-408, Glu-416, Lys-422, Glu-423, Asp-425, Arg-431, and Glu-438, which were replaced by Ala, are shown as sticks. c, amino acid sequences of the middle domain motif-1 of ClpB/Hsp104 from T. thermophilus (TClpB), E. coli (EClpB), S. cerevisiae (ScHsp104), and Chaetomium thermophilum (CtHsp104) are aligned. The positively and negatively charged conserved residues, which were replaced by Ala, are shown in blue and red, respectively.

Figure 2.

Effects of alanine substitutions of conserved charged residues in TClpB middle domain motif-1. a, ATPase activities of TClpB mutants, in the absence (gray bar) or the presence (open bar) of 0.1 mg/ml κ-casein, are shown as turnover number per monomer. The measurements were performed in the presence of 3 mm ATP at 55 °C using an ATP-regeneration system. b, disaggregation activities of TClpB mutants. α-Glucosidase (final concentration, 0.2 μm monomer) was heat-aggregated by incubation at 73 °C for 10 min in the presence of 3 mm ATP. TDnaK (0.6 μm), TDnaJ (0.2 μm), TGrpE (0.1 μm dimer), and TClpB mutants (0.05 μm hexamers) were added to the reaction mixture, and the mixture was subsequently incubated at 55 °C for 90 min. After incubation, the recovered enzymatic activities were measured and are shown as a percentage of the activities before heat aggregation. c, chaperone activities of TClpB mutants for the reactivation of soluble, denatured proteins were measured by using α-glucosidase as a substrate. The experimental procedures were essentially the same as for b, except that the TDnaK, TDnaJ, and TGrpE were added prior to the heat treatment, and the substrate was denatured but not aggregated. a–c, error bars represent standard deviations of three or more independent experiments.

Figure 3.

Essential charged residues in the middle domain motif-1 involved in nucleotide-dependent inter-subunit interactions. To form covalent bonds between the essential charged residues in motif-1 and the closely positioned C–H bond, BPM-labeled TClpB mutants were UV-irradiated at 365 nm for 90 min in the absence or presence of 3 mm nucleotides. The cross-linked samples were analyzed by SDS-PAGE (5% polyacrylamide). a, gel images; b, intensities of bands corresponding to 1–5- and over 6-mers of E401C-BPM and D404C-BPM, with or without Walker-A mutations are shown. b, error bars represent standard deviations of three or more independent experiments.

Figure 4.

Structural models of TClpB. The structural models of the TClpB hexamer were constructed by homology modeling using cryo-EM structures of EClpB with or without casein (PDB codes 5og1 and 5ofo) as templates. a and b, interfaces between AAA1 (light blue) and middle domain motif-1 (yellow) of neighboring subunits. AAA1 and motif-1 correspond to chains C and D (the middle domain is in the tilted conformation) of 5og1 (a), and chains B and C (the middle domain is in the horizontal conformation) of 5ofo (b). Amino acid residues, Glu-401, Asp-404, Asp-176, Glu-177, Arg-180, Arg-181, Arg-214, Asp-219, and Glu-222, which were replaced by Ala, are shown as sticks. c, amino acid sequences of a part of AAA1 of ClpB/Hsp104 from T. thermophilus (TClpB), E. coli (EClpB), S. cerevisiae (ScHsp104), and C. thermophilum (CtHsp104) were aligned. The positively and negatively charged conserved residues, which were replaced by Ala, are shown in blue and red, respectively. d and e, TClpB structural model constructed based on chain D of 5og1 (d) and chain C of 5ofo (e). Amino acid residues Glu-401 and Arg-532 are shown as sticks. f, amino acid sequences of a part of AAA1 of TClpB, EClpB, ScHsp104, and CtHsp104 were aligned. Positively and negatively charged conserved residues are shown in blue and red, respectively.

Figure 5.

Effects of alanine substitutions of the charged residues in the region of AAA1 that potentially interact with Glu-401 and Asp-404. a, ATPase activities of TClpB mutants in the absence (gray bar) or presence (open bar) of 0.1 mg/ml κ-casein are shown. b, disaggregation activities of TClpB mutants were measured using α-glucosidase as a substrate. c, chaperone activities of TClpB mutants for the reactivation of soluble, denatured proteins were measured by using α-glucosidase as a substrate. Experimental procedures for a–c were the same as for Fig. 2, a–c. Error bars represent standard deviations of three or more independent experiments.

Figure 6.

Effects of charge-inversion of potentially interacting pairs of amino acid residues. a and b, ATPase activities of TClpB mutants having charge-inversion mutations, in the absence (gray bar) or presence (open bar) of 0.1 mg/ml κ-casein, are shown. c and d, disaggregation activities of TClpB mutants were measured using α-glucosidase as a substrate. e and f, chaperone activities of TClpB mutants for the reactivation of soluble, denatured proteins were measured using α-glucosidase as a substrate. a, c, and e, activities of TClpB mutants having a single charge-inversion mutation. b, d, and f, activities of TClpB mutants having combined charge-inversion mutations. a–f, experimental procedures were the same as Fig. 2, a–c, and error bars represent standard deviations of three or more independent experiments.

Figure 7.

Schematic model of ATP-dependent changes in interactions between the middle domain motif-1 and AAA1 of neighboring subunits. a, Glu-401, but not Asp-404, is located near the loop, including Asp-219 and Glu-222. b, ATP binding to AAA1 causes sliding and/or rotational motion of the middle domain in which the Asp-404 moves close to the loop instead of Glu-401. c, structural change enables Glu-401 to interact with Arg-532. This interaction shifts the equilibrium of the middle domain conformation between horizontal and tilted to tilted conformation. The shift would support DnaK-induced middle domain tilting (i.e. ClpB activation). In the activated form, the Arg-180–Asp-404 electrostatic interaction might contribute to inter-subunit signal transduction that is essential for the chaperone activities of ClpB.