Structural characterization of the thermal unfolding pathway of human VEGFR1 D2 domain

Abstract

Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an important complement to protein structure and function, crucial to determine the structural basis of protein misfolding. In this context, thermal unfolding studies represent a useful tool to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein. Here, we report the thermal folding/unfolding pathway of VEGFR1D2, a member of the immunoglobulin superfamily by means of a high-resolution thermodynamic approach that combines differential scanning calorimetry with atomic-level unfolding monitored by NMR. We show how VEGFR1D2 folding is driven by an oxidatively induced disulfide pairing: the key event in the achievement of its functional structure is the formation of a small hydrophobic core that surrounds a disulfide bridge. Such a 'folding nucleus' induces the cooperative transition to the properly folded conformation supporting the hypothesis that a disulfide bond can act as a folding nucleus that eases the folding process.

Keywords: DSC; NMR; VEGF; disulfide bond; thermal unfolding.

Fig. 1

(A) Heat capacity curves of 35 µm VEGFR1D2 in buffer (20 mm Tris‐HCl, pH = 7.0, 120 mm NaCl) heating rate 1 K·min⁻¹ (blue circles). Red dashed lines represent baselines estimated as reported in the text. (B) Excess molar heat capacity curves of VEGFR1D2 obtained from the calorimetric scans shown in the left panel. Blue circles, black lines, and red lines represent the experimental data, the obtained fits are their deconvoluted components, respectively.

Fig. 2

(A) ESI‐ToF MS analysis of VEGFR1D2 alkylated at room temperature (RT) after incubation at 353 K in presence and absence of DTT. (B) Untreated VEGFR1D2; (B) VEGFR1D2 treated at 353 K for 40 min and alkylated with iodoacetamide at RT for 30 min; and (C) VEGFR1D2 treated at 353 K for 40 min in presence of DTT and alkylated with iodoacetamide at RT for 30 min. In the insets are reported the deconvoluted mass spectra showing the experimental average mass values of the species. VEGFR1D2 (*), MW_th (av): 11851.688 Da; reduced VEGFR1D2 (¥), MW_th (av): 11853.688; monoalkylated VEGFR1D2 (#), MW_th (av): 11910.738 Da; dialkylated VEGFR1D2 ($), MW_th (av): 11967.788 Da.

Fig. 3

ToF MS analysis of VEGFR1D2 incubated and alkylated at 353 K, in presence and absence of DTT. (A) Deconvoluted ESI‐ToF mass spectrum of VEGFR1D2 treated at 353 K for 40 min and alkylated at 353 K for 30 min with iodoacetamide. (†): VEGFR1D2 with the intact disulfide bridge and unspecifically dialkylated on residues other than Cys (MW_th (av): 11965.788 Da); (‡): VEGFR1D2 unspecifically trialkylated on residues other than Cys (MW_th (av): 12022.838 Da). (B) Deconvoluted ESI‐ToF mass spectrum of VEGFR1D2 treated at 353 K for 40 min in presence of DTT and alkylated with iodoacetamide at 353 K for 30 min. (#): VEGFR1D2 with reduced cysteines and monoalkylated (MW_th (av): 11910.738 Da); (*): VEGFR1D2 with reduced cysteines and dialkylated (MW_th (av): 11967.788 Da); ($): VEGFR1D2 with reduced cysteines and trialkylated (MW_th (av): 12024.838 Da); (§): VEGFR1D2 with reduced cysteines and tetralkylated (MW_th (av): 12081.888 Da).

Fig. 4

(A) Overlay of [¹H‐¹⁵N] HSQC spectra of VEGFR1D2 up to the last temperature (323 K) at which the signals are observed; (B) Overlay of 2D [¹H, ¹⁵N] HSQC spectra of VEGFR1D2 at 298 K (red) and 353 K (blue).

Fig. 5

(A) Bar graphs of the average combined chemical shift differences (ΔHN_av) as a function of residue number. The mean value (plus the standard deviation) (ΔHN_av = 0.10 ppm) is shown as a continuous line. The secondary structure elements are also indicated. (B) CSP mapping onto the representative conformer of the NMR structure of VEGFR1D2 shown as ribbon drawing. Residues for which ΔHNav > 0.10 ppm are shown in magenta. Disulfide bridge between C158 and C207 is highlighted in yellow. (C) Bar graphs of the intensity difference (I ₀–I/I) as a function of residue number. The mean value (I ₀–I/I = 0.55 ppm) is shown as a broken line. The secondary structure elements are also indicated. (D) Intensity difference mapping onto the representative conformer of the NMR structure of VEGFR1D2 shown as ribbon drawing. Residues for which I ₀–I/I > 0.55 ppm mare shown in red. Disulfide bridge between C158 and C207 is highlighted in yellow. Structures figures were created with the program Pymol.

Fig. 6

ESI‐ToF MS analysis of VEGFR1D2. (A) VEGFR1D2 (20 μm) was incubated in 20 mm phosphate buffer, 10 mm TCEP, pH 7.0, for 24 h at room temperature. The protein solution was centrifuged and the insoluble fraction was solubilized with 8 M guanidinium hydrochloride and analyzed by LC‐MS ESI ToF. The mass spectrum of the protein peak is reported; the inset shows the deconvoluted mass spectrum. For comparison, the mass spectrum of VEGFR1D2 is also reported (B).