Fig 1.
Mortalin production and isolation.
Recombinant human mortalin (pET28a::mtHsp70) was co-expressed with recombinant hHep1 (pET23a::hHep1) in E. coli BL21(DE3) cells. A) SDS-PAGE of the produced and purified recombinant mortalin. 1) MM markers in kDa (left); 2) non-induced bacterial pellet; 3) induced bacterial pellet; 4) pellet of lysed cells; 5) supernatant of lysed cells; 6) fraction obtained from Ni2+ affinity chromatography; and 7) preparative SEC fraction. The final purity of mortalin was higher than 95%. B) aSEC profile of mortalin after Ni2+ affinity chromatography (red line), which showed that mortalin was eluted into four fractions (see text for details). The monomeric fraction (4) was immediately reloaded into the aSEC column and eluted as a monomer (blue line). The standard protein mixture profile is represented by the black line, and the MM of each protein is shown. The vertical dashed line marks the monomeric mortalin elution volume.
Fig 2.
Mortalin was produced in its folded state.
A) The CD spectrum shows that mortalin was obtained with a secondary structure composed of both α-helices and β-sheets. The secondary structure content, which was estimated by the CDNN deconvolution, is depicted into the figure (error < 5%). B) The intrinsic fluorescence emission (excitation λ at 295 nm) spectrum of mortalin had a λmax value of 337 ± 1 nm and a <λ> value of 346.1 ± 0.2 nm, indicating that the single Trp residue was at least partially buried. In the presence of 8 mol.L-1 urea, the spectrum was quenched and suffered a red shift (λmax of 349 ± 1 nm and <λ> of 351.8 ± 0.2 nm), suggesting unfolding. Inset: normalized spectra. C) Mortalin CD spectra in the presence of the indicated ligands. D) Intrinsic fluorescence emission (excitation λ at 295 nm) spectra of mortalin in the presence of the indicated ligands, which led to a suppression of the fluorescence emission intensity and to a slightly blue shift in the spectra, as shown in the normalized fluorescence spectra (inset). These results suggested that mortalin was expressed in both its folded and functional state. Moreover, the presence of adenosine nucleotides and Mg2+ ions led to slight conformational changes in the mortalin structure.
Fig 3.
Monomeric mortalin was slightly elongated in solution.
A) Estimation of the mortalin Rs based on the aSEC data presented in Fig. 1B. The graph depicts the Rs of standard globular proteins as a function of the partial coefficient kav, which yielded an Rs value for mortalin of 35 ± 2 Å. B) Sedimentation velocity data resulting from the c(S) distribution of mortalin, which behaved mainly as a monomeric species of 76 ± 4 kDa with ƒ/ƒ0 of approximately 1.36 ± 0.01 under the tested conditions (see Materials and Methods section). Inset: Determination of the s020,w of mortalin through the dependence of s20,w on the protein concentration. Mortalin had a s020,w value of 4.8 ± 0.1 S (Table 1).
Table 1.
Summary of the hydrodynamic and structural data of mortalin.
Fig 4.
Mortalin has higher ATPase activity than Hsp70–1A.
A) Mortalin (2.50 μmol.L-1) and Hsp70–1A (2.25 μmol.L-1) were incubated with ATP (0–2 mmol.L-1) for 90 min at 37°C, and the Pi released as a result of ATP hydrolysis was quantified. The data were treated through Michaelis-Menten fitting for determination of the kinetic parameters, which are presented in the Figure and Table 2. The results suggested that both mortalin and Hsp70–1A exhibit low ATPase activity. Despite these findings, based on the kcat value, mortalin presented higher ATPase activity than Hsp70–1A, although the KM values of both are similar. B) Relative ATPase activity stimulation. Effect of the NR peptide titration on the basal ATPase activity of mortalin and Hsp70–1A at 1 mmol.L-1 ATP.
Table 2.
Kinetic constants determined for human mortalin and human Hsp70–1A compared with those of homologous Hsp70.
Fig 5.
Mortalin interacts with ADP and ATP in a micromolar dissociation constant range.
The mortalin interaction with ADP (A) and ATP (B) in the presence of Mg2+ was tested by ITC, suggesting KDs values of approximately 2.2 ± 0.1 μmol.L-1 and 1.1 ± 0.1 μmol.L-1, respectively. Moreover, the ITC data suggested that the interaction was directed by both enthalpy and entropy. Upper panel: The heat released at each ADP or ATP titration is presented by the negative peaks. The red line represents the baseline. Lower panel: The ΔHapp values were calculated by the integrated area of each ADP or ATP titration peak of the upper panel and plotted against the ADP/mortalin molar ratio. The red line represents the fit obtained by the one-site-binding model provided by the Origin program supplied with the ITC device in both cases. The fitting parameters are shown.
Table 3.
Summary of the Tm transitions determined to mortalin by CD222 nm in the presence of adenosine nucleotides and/or Mg2+.
Fig 6.
Mortalin has an elongated shape in solution.
A) Experimental mortalin SAXS curve (open circles) suggesting that it behaved as a monodisperse system, as was confirmed by the evaluation of the Guinier region of the curve (inset). The GNOM fit is represented by a black line. Based on Guinier’ law (red line—inset), mortalin had a Rg value of 36 ± 2 Å (see text for details). B) The SAXS data were used to generate the p(r) distribution curve, which indicated that mortalin has a prolate shape and a Dmax value of 130 ± 10 Å.
Fig 7.
The mortalin SAXS curve was used by calculating 20 low-resolution ab initio models using the DAMMIN software, and these were merged using the DAMAVER package. The result is the final ab initio model presented in several orientations (A). Manual superposition of the crystallographic structures of the mortalin NBD (PDB acc. no. 4KBO—magenta) and PBD of the E. coli DnaK (PDB acc. no. 1DKX—blue) into the ab initio model (B). Manual superposition of the crystallographic structure of the full length E. coli DnaK (PDB acc. n. 4B9Q—blue) into the mortalin ab initio model (C). Figures generated by the UCSF Chimera software (version 1.9).
Fig 8.
Mortalin is composed of at least two domains with different stabilities.
A) The thermal-induced unfolding of mortalin followed by CD222 nm presented two well-defined transitions with Tm values centered at 40 and 73°C; however, mortalin did not unfold completely (see text for details). B) Thermal-induced unfolding of mortalin followed by intrinsic fluorescence emission and represented as the <λ>-signal showing that mortalin suffered two blue-shift transitions with Tm values of approximately 43 and 77°C. Hsp70–1A presented two red-shift transitions with Tm values at 50.5 and 70.8°C. N-acetyl tryptophanamide at the same buffer conditions was used, as a control, and no transitions were observed at the <λ>-signal (351.5 ± 0.2 nm). The blue-shift transition suggested that mortalin associated or aggregated in the thermal-induced unfolding experiments (see text for details).