Molecular chaperones, essentially including heat shock proteins (Hsps), are recognized as a protein quality control machinery that operates to ensure cellular proteostasis (
The Hsp40 family proteins generally share several characteristic domains/motifs, including a J-domain (JD), a glycine/phenylalanine-rich region (GF-motif), a cysteine repeat region containing a zinc-finger-like motif, and substrate-binding domains. Particularly, the highly conserved JD is recognized as a principle region of Hsp40 for its interaction with Hsp70, whereas the other regions are presumed to facilitate the targeting of Hsp70 chaperones to specific substrates (11). Both the overall primary sequences and the composition and organization of structural domains vary in individual Hsp40 proteins. For instance, in humans, Hsp40s comprise a variety of subfamilies with more than 40 different members, which vary in their substrate selectivity and individual functions in cells. Based on the structural components, the Hsp40 family is divided into three groups: DNAJA, DNAJB, and DNAJC (8). The former two group members, including DnaJ in bacteria (4) commonly share the JD at their N-terminus (Fig. 1A), whereas the JD is present elsewhere in the DNAJC proteins (12). The N-terminal JD of the DNAJA proteins is followed by a GF-motif, two homologous β-barrel domains containing a zinc-finger-like region in the first sequence, and a C-terminal dimerization domain. The DNAJA members are also subdivided into four subfamilies, from DNAJA1 to DNAJA4. The structural components of the DNAJB group are similar to those of DNAJA, but lack the zinc-finger-like region.
Tumorous imaginal disc 1 (Tid1) was identified as a tumor suppressor that is involved in diverse cellular processes, in both the cytosol and mitochondria, that include imaginal discs differentiation, T cell development, and mitochondrial apoptosis signaling (13-16). However, as Tid1 has an N-terminal signal peptide for mitochondrial targeting (Fig. 1A), the protein localizes to the mitochondria and functions as an Hsp40 that participates in the co-chaperone activity of mortalin, the mitochondrial Hsp70 (17, 18). Mortalin and Tid1 constitute the only Hsp70/Hsp40 chaperone system in mitochondria, and in particular, the regulation of apoptosis in mitochondria is initiated by the interaction of Tid1 with mortalin via its JD.
As the domain organization of Tid1 is comparable to that of the DNAJA2-subfamily proteins, except for the presence of the N-terminal mitochondrial targeting sequence, Tid1 is classified as in the DNAJA3 subfamily of Hsp40 (Fig. 1A). However, whether the detailed structure and interdomain interactions, which would provide a molecular basis for substrate selectivity and Hsp70 interaction, are consistent with those of other related proteins remains to be investigated. In particular, it was recently elucidated that the interdomain interaction of JD with the GF-motif differs between the DNAJA2 and DNAJB1 subfamilies (19). Therefore, in the present study, we aimed to determine the three-dimensional structure of Tid1-JD and its interaction with the GF-motif in solution using nuclear magnetic resonance (NMR) spectroscopy.
A recent report by Faust
The determined NMR structure of the Tid1 JD is shown in Fig. 2, and the structural statistics are shown in Supplementary Table 1. The root mean square deviation (RMSD) of the 20 lowest-energy structures from their average structure was 0.39 ± 0.08 Å for backbone and 0.97 ± 0.10 Å for all heavy atoms, indicating a good convergence of the ensemble structure (Fig. 2A). Overall, the structure showed a typical JD fold composed of four α-helices: α1 (residues 94-99), α2 (105-118), α3 (131-145), and α4 (149-157). The HPD motif, which is known to contain key residues for the interaction of a JD in HSP40 with its cognate Hsp70, adopted a flexible loop conformation between α2 and α3 (Fig. 2B). Although no JD structure was available for DNAJA2, the overall conformation of the Tid1 JD showed a well-matched structural superimposition on the known DNAJB1 JDGF structure (Fig. 2C), as expected from the sequence similarity (Fig. 1B). The RMSD between the JD structures of Tid1 and DNAJB1 for superposition of all Cα atom pairs in the 89-159 region of Tid1 was 2.236. Therefore, we further investigated whether the GF-motif interacts with JD in Tid1, as in DNAJB1.
The 2D [1H-15N]HSQC spectrum of JD showed an overall inconsistency with that of JDGF (Fig. 3A), implying alteration of the chemical environment of the JD, probably due to direct interaction with the GF-motif. The most significantly affected residues, which were defined by weighted chemical shift perturbations (CSPs) exceeding 0.45 ppm (Fig. 3B), included Q115, K118, H121, and A139. These amino acids were located mainly on the surface of the α2, α3, and α2-α3 loop regions (Fig. 3C), which mediate the Hsp70 interaction in the JD (19). In particular, the significant CSPs at H121 and T124 suggest that the HPD motif (Fig. 2B) is likely blocked by the GF-motif interaction.
It was confirmed by comparing NMR relaxation dynamics between the JD and JDGF that the overall CSPs are not attributable to a global conformational change of JD (Fig. 4). As expected from the larger size, which increases the rotational correlation time, JDGF showed higher values of longitudinal relaxation time (T1) and lower values of transverse relaxation time (T2) than JD in individual residues. However, the overall patterns of the T1 and T2 traces along the sequence (
DNAJB proteins constitute a major class of Hsp40 proteins in humans. The aforementioned research by Faust
In the present study, we found that the solution structure of the Tid1 JD was consistent with the known JD structure of DNAJB1. In addition, as the GF-motif stably binds to JD in Tid1, as in DNAJB1, it is postulated that the JD interaction with Hsp70 is intrinsically blocked and require an allosteric interaction with Hsp70 to release that inhibition. The structural resemblance of Tid1 to DNAJB1 suggests that the mitochondrial Hsp40/Hsp70 chaperone system may have evolved to employ the specific functional mode of the major class of Hsp40 and DNAJB proteins.
NMR chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank under the following accession codes: 36474, 26322 for Tid1-JD and Tid1-JDGF, respectively. The structure of Tid1-JD has been deposited to the Protein Data Bank (PDB) under accession code 7X89.
Recombinant proteins corresponding to the Tid1 JD (residues 89-159) and its extended form, JDGF (residues 89-192), which encompasses both the JD and GF-motif, were prepared for NMR, as described previously (20). Briefly, DNA fragments encoding the JD and JDGF were subcloned from the cDNA of human Tid1 into the pCold-I vector plasmids. The
The isotope [13C/15N]-enriched protein samples for NMR were dissolved in 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 1 mM dithiothreitol, and 7 % (v/v) D2O. Two-dimensional HSQC spectra and three-dimensional (3D) NMR data were recorded at 25°C on a Bruker 800 or 900 MHz NMR spectrometer equipped with a cryoprobe. The measured NMR spectra were processed using the NMRPipe/NMRDraw software (23) and analyzed with the NMRView program (24). Based on the previously achieved backbone NMR assignments (20), the side-chain assignments of the JD were performed using 3D HBHA(CO)NH, HCCH-COSY, and 15N-HCCH-TOCSY spectra. The combined chemical shift perturbations were represented by [(ΔδNH2 + ΔδN2/25)/2]1/2, where ΔδNH and ΔδN are the chemical shift changes of backbone amide proton (NH) and amide (N), respectively.
Distance restraints for structure calculation were obtained from 3D [15N]- and [13C]-edited NOESY-HSQC NMR spectra, of which NOE cross-peaks were assigned by the auto-assignment function of CYANA 2.1 (25). Dihedral ϕ and φ angle restraints were derived from the prediction of TALOS+ (26) using the previously assigned backbone NMR chemical shifts (20). Initial structures of the Tid1 JD were generated with CYANA 2.1, followed by refinements with the statistical torsion angle potential energy functions (27) using the Charmm program (28). The final 20 structures with the lowest energy were validated by three types of scores:: DOPE (29), normalized DOPE, and dDFIRE (30). Ramachandran plot appearance was measured using MolProbity (31).
The longitudinal relaxation rate (R1) and the transverse relaxation rate (R2) of backbone 15N nuclei, as well as the heteronuclear NOEs between amide 15N and 1H nuclei were assessed using established HSQC-based NMR pulse sequences. Relaxation experiments were performed at 900 MHz (21.2 Tesla). T1 values were measured from 1H-15N correlation spectra recorded with relaxation evolution delays of 50, 100, 200, 300, 500, 700, 900, and 1200 ms. A 3 s delay was used between scans. T2 values were measured from 1H-15N correlation spectra recorded using relaxation evolution delays of 16.96, 33.92, 67.84, 101.76, 135.68, 169.6, 203.52, and 254.4 ms. A delay of 2 s was used between scans. The steady-state 1H-15N heteronuclear NOE values were determined from the peak ratios observed between the two spectra, which were collected with or without pre-saturation of the proton dimension. A delay of 3 s was used between the scans.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1A5A2015541, 2019R1F1A1057427 and 2019R1A2C1004883) and by ”Regional Inovation Strategy (RIS)“ through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021RIS-001). The use of NMR was supported by the Korea Basic Science Institute under the R&D program (Project No. C140440), supervised by the Ministry of Science and ICT.
The authors have no conflicting interests.