Equilibrium unfolding of the PDZ domain of β2-syntrophin

Abstract

β2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic β-cells. It contains a PDZ domain (β2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of β2-syntrophin allosterically regulates the affinity of β2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of β2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, β2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, T(m) and T(max) denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 and the results of molecular dynamics simulations indicate that this behavior of β2S-PDZ might be related to its charge distribution. All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and the regulation of insulin secretion.

Figure 1

CD analysis of β2S-PDZ. (A) Far-UV spectra at 20°C (black line), 4°C (gray line), and 90°C (dashed line). (B) Mean molar ellipticity at 220 nm (circles). A two-state fit to the data (Eqs. 1 and 3) is shown as a solid line.

Figure 2

Thermal denaturations of β2S-PDZ monitored by ellipticity at 220 nm. Two-state fits and baselines are shown as heavy solid and dashed lines, respectively. The fit procedure and considerations for the baselines are explained in the text. (A) Transitions at different pH (solid lines). (B) Thermal denaturations of β2S-PDZ at different urea concentrations (solid lines) of (bottom to top) 0, 0.4, 0.8, 1.2, 1.6, 2, and 7.5 M. In both panels, two-state fits and baselines are shown as heavy solid and dashed lines, respectively. The fit procedure and considerations for the baselines are explained in the text.

Figure 3

Thermodynamic parameters obtained from two-state fitting of the melting curves at different urea concentrations. ΔH_Tm (left), T_m (middle, black), T_max (middle, gray), and ΔC_P (right).

Figure 4

Spectral analysis of the temperature transitions. (A) CD spectra taken at 2° intervals. (B) Normalized temperature-dependent CD signal monitored at 204 nm (black solid circles), 208 nm (gray open circles), and 220 nm (black open circles) obtained from the spectra in A. (C) Amplitude of the two significant components, A (gray line) and B (black line), obtained by SVD of the spectra in A and weighted by their singular values. (D) Melting curve obtained by addition of the variation of the principal components. Calculations are described in the text.

Figure 5

Structural alignment of a low-redundancy set of PDZ domains. Boxes indicate the position of canonical structure elements. Triangles point to basic amino acids relevant for function or present only in β2S-PDZ. Only basic amino acids were highlighted: the most conserved residues appear in black and the less conserved in gray background. The left column lists the pdb codes of each structure included in the alignment: β2S-PDZ (2vrf), sixth PDZ and PDZ12 domains of glutamate-receptor-interacting protein 1 (1n7e and 2qt5, respectively), fourth PDZ domain of 2 scribble protein (1uju), second and third PDZ domains of disc large homolog 2, PSD-93 (2byg and 2he2, respectively), Golgi-associated PDZ domain (2dc2), first and second PDZ domains of DLG3 (2fe5 and 2i1n, respectively), and PDZ2 of PTP-BL (1gm1).

Figure 6

Ribbon representation of four PDZ-domain structures. PDZ2 of PTP-BL (1gm1), second and third PDZ domains of disc large homolog 2, PSD-93 (2byg and 2he2, respectively) and β2S-PDZ (2vrf). In the foreplane are α-helix B and the protein-binding pocket. The side chains of charged residues are shown in all four structures. The amino acids in the characteristic cluster of arginine and lysine residues in β2S-PDZ are labeled.

Figure 7

Cβ contact maps of four PDZ domains. The circles correspond to positive repulsive contacts (black solid circles), negative repulsive contacts (gray solid circles), attractive contacts (black open circles), and nonionic contacts (gray open circles). PDB codes are indicated in each panel and correspond to the sixth PDZ domain of glutamate-receptor interacting protein 1 (1n7e), PDZ2 of PTP-BL (1gm1), the third PDZ domain of postsynaptic density protein PSD-93 (2he2), and β2S-PDZ (2vrf).

Figure 8

Energy diagrams of molecular dynamics simulations using structure-based force fields. The energy (F) of each conformation is represented as a function of the fraction of native contacts (Q). The black line was obtained using a plain structure-based force field, whereas the gray line is the result of including the electrostatic effects in the force field.