HspB2/myotonic dystrophy protein kinase binding protein (MKBP) as a novel molecular chaperone: structural and functional aspects

Abstract

The small heat shock protein, human HspB2, also known as Myotonic Dystrophy Kinase Binding Protein (MKBP), specifically associates with and activates Myotonic Dystrophy Protein Kinase (DMPK), a serine/threonine protein kinase that plays an important role in maintaining muscle structure and function. The structure and function of HspB2 are not well understood. We have cloned and expressed the protein in E.coli and purified it to homogeneity. Far-UV circular dichroic spectrum of the recombinant HspB2 shows a β-sheet structure. Fluorescence spectroscopic studies show that the sole tryptophan residue at the 130(th) position is almost completely solvent-exposed. Bis-ANS binding shows that though HspB2 exhibits accessible hydrophobic surfaces, it is significantly less than that exhibited by another well characterized small HSP, αB-crystallin. Sedimentation velocity measurements show that the protein exhibits concentration-dependent oligomerization. Fluorescence resonance energy transfer study shows that HspB2 oligomers exchange subunits. Interestingly, HspB2 exhibits target protein-dependent chaperone-like activity: it exhibits significant chaperone-like activity towards dithiothreitol (DTT)-induced aggregation of insulin and heat-induced aggregation of alcohol dehydrogenase, but only partially prevents the heat-induced aggregation of citrate synthase, co-precipitating with the target protein. It also significantly prevents the ordered amyloid fibril formation of α-synuclein. Thus, our study, for the first time, provides biophysical characterization on the structural aspects of HspB2, and shows that it exhibits target protein-dependent chaperone-like activity.

Figure 1. SDS-PAGE pattern showing purification of HspB2 from the soluble fraction of E.coli BL21(DE3) cells over-expressing human HspB2.

Lane 1, whole cell lysate; Lane 2, soluble fraction; Lane 3, insoluble fraction; respectively, Lane 4, HspB2 precipitated by 20% saturated ammonium sulfate; and Lane 5, HspB2 purified by Phenyl Sepharose chromatography.

Figure 2. Far-UV (a) and near-UV (b) CD spectra of HspB2.

Solid line, human HspB2; dotted line, αB-crystallin. The far- and near-UV CD spectra of the proteins, were recorded using 0.1 cm and 1.0 cm path length cuvettes respectively. [θ]MRW is mean residue ellipticity.

Figure 3

(a) Intrinsic fluorescence spectrum of HspB2. The excitation wavelength was 295 nm. The excitation and the emission band passes were set at 2.5 nm. The concentration of the proteins was 0.2 mg/ml. Fluorescence intensity is represented in arbitrary units. (b) Lehrer plot of tryptophan fluorescence quenching of HspB2 by KI. Fluorescence was monitored after sequential addition of KI to a solution containing 0.2 mg/ml HspB2. Insert shows the decrease in fluorescence intensity upon sequential addition of KI.

Figure 4. Sedimentation velocity profile of HspB2.

Distribution of sedimentation coefficient of different oligomeric species changes with increasing concentration of HspB2. Panels A, B and C correspond to 0.125, 1 and 3 mg/ml of HSPB2. The numbers depicted above the peaks correspond to molecular masses (in kDa) of the oligomeric species.

Figure 5. Bis-ANS binding to HspB2.

(a) Fluorescence spectra of bis-ANS alone (dotted line) and bis-ANS bound to human HspB2 (solid line). Bis-ANS was added to the final concentration of 10 µM and the spectra were recorded using an excitation wavelength of 390 nm with the excitation and emission band passes set at 2.5 nm. (b) Bis-ANS titration of human HspB2. Bis-ANS fluorescence was measured as described above after sequential additions of bis-ANS to the protein solutions. (-•-) αB-crystallin, (-▴-) HspB2.

Figure 6. Subunit exchange studies of HspB2 at different temperatures.

Subunit exchange at (a) 37°C (b) 4°C. The fluorescence spectra immediately after mixing AIAS and LYI labeled HspB2 in equimolar ratios (dotted line) and after 15 min (solid line) are represented. A decrease in AIAS fluorescence intensity with concomitant increase in LYI fluorescence intensity indicative of subunit exchange is seen when the mixture is incubated at 37°C. No such change was seen at 4°C. The rate of subunit exchange at 37°C (•), 20°C (▴) are compared (c). The rates of subunit exchange at these two temperatures are similar.

Figure 7. Chaperone-like activity of human HspB2 towards DTT-induced aggregation of insulin.

(a) Aggregation profile of 0.2 mg/ml insulin alone (1); and in the presence of 1∶0.0625, (2); 1∶0.125, (3); 1∶0.25, (4); and 1∶0.5, (5) ratios of insulin to HspB2 (w/w). (b) The percentage protection of DTT-induced aggregation of insulin by HspB2 as a function of weight ratio of chaperone to target protein.

Figure 8. Effect of HspB2 on the thermal aggregation of citrate synthase (CS) at 43°C.

(a) Aggregation profile of 25 µg/ml CS alone (1) and in the presence of 1∶4, (2); 1∶2, (3); 1∶1, (4) 1∶0.5, (5) and 1∶0.25, (6) ratios of CS to HspB2 (w/w). (b) The apparent percentage protection calculated from light scattering profile as a function of the weight ratio of CS to HspB2. (c) The SDS-PAGE pattern of soluble (S) and precipitate (P) fractions of mixtures of various citrate synthase to HspB2 ratios used in the chaperone assays. The soluble fraction and the precipitate were obtained by centrifugation at the end of the chaperone assay. (d) Effect of HspB2 on the thermal aggregation of yeast alcohol dehydrogenase (ADH) at 48°C. Aggregation profile of 0.2 mg/ml ADH alone (1) and in the presence of 1∶0.1, (2); 1∶0.2 (3); 1∶0.5, (4); 1∶2 (5); ratios of alcohol dehydrogenase to HspB2 (w/w). (e) the percentage protection calculated from light scattering profile as a function of the weight ratio of ADH to HspB2. (f) SDS-PAGE pattern of soluble (S) and precipitate (P) fractions of mixtures of various ADH to HspB2 ratios used in the chaperone assays. The soluble fraction and the precipitate were obtained by centrifugation at the end of the chaperone assay.

Figure 9. Prevention of α-synuclein amyloid fibril formation by HspB2.

(a) Amyloid fibril formation of α-synuclein in the absence (-○-) and in the presence of HspB2 at α-synuclein to HspB2 ratio of 1∶1.5 (-•-) monitored by ThT fluorescence. HspB2 incubated with α-synuclein seeds (-▴-), or HspB2 alone (-Δ-) showed negligible ThT fluorescence. (b) Percentage prevention of α-synuclein amyloid fibril growth in the presence of various concentrations of HspB2. Error bars for 7 sets of experimental data are shown.

Figure 10. Temperature-induced changes in the conformation and chaperone property of HspB2.

(A) far UV-CD spectra of HspB2 at 25°C (1), 45°C (2) and 55°C (3). (B) The change in the mean residue mass ellipticity ([θ]MRM) at 214nm as a function of temperature. (C) Near UV-CD spectra of HspB2 at 25°C (1), 45°C (2) and 55°C (3). (D) The change in the [θ]MRM at 286 nm as a function of temperature. (E) The changes in the fluorescence polarization of AIAS-labelled HspB2 with increasing temperature. (F) % Protection offered by HspB2 to the DTT-induced aggregation of insulin at different temperatures.