A novel method of production and biophysical characterization of the catalytic domain of yeast oligosaccharyl transferase

Abstract

Oligosaccharyl transferase (OT) is a multisubunit enzyme that catalyzes N-linked glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum. In the case of Saccharomyces cerevisiae, OT is composed of nine integral membrane protein subunits. Defects in N-linked glycosylation cause a series of disorders known as congenital disorders of glycosylation (CDG). The C-terminal domain of the Stt3p subunit has been reported to contain the acceptor protein recognition site and/or catalytic site. We report here the subcloning, overexpression, and a robust but novel method of production of the pure C-terminal domain of Stt3p at 60-70 mg/L in Escherichia coli. CD spectra indicate that the C-terminal Stt3p is highly helical and has a stable tertiary structure in SDS micelles. The well-dispersed two-dimensional (1)H-(15)N HSQC spectrum in SDS micelles indicates that it is feasible to determine the atomic structure by NMR. The effect of the conserved D518E mutation on the conformation of the C-terminal Stt3p is particularly interesting. The replacement of a key residue, Asp(518), located within the WWDYG signature motif (residues 516-520), led to a distinct tertiary structure, even though both proteins have similar overall secondary structures, as demonstrated by CD, fluorescence and NMR spectroscopies. This observation strongly suggests that Asp(518) plays a critical structural role, in addition to the previously proposed catalytic role. Moreover, the activity of the protein was confirmed by saturation transfer difference and nuclear magnetic resonance titration studies.

Figure 1

Coomassie-stained SDS-PAGE of samples from a typical C-terminal Stt3p expression and purification run. The mobility of the His-tagged C-terminal Stt3p in the SDS-PAGE gel is compatible with its molecular mass (31.4 kDa). Lane 1, before induction; Lane 2, 4 hours after induction with 0.5mM IPTG; Lane 3, inclusion body; Lane 4, protein molecular weight markers; Lane 5-8, protein purified by “SDS Elution” where elution fractions: 1, 2, 3 and 4 are shown.

Figure 2

MALDI-TOF analysis of molecular mass of the purified His-tagged C-terminal domain of Stt3p. The mass spectrum of the purified protein showed a molecular ion at m/z 31413.1, which is in accordance with the calculated value (31422.3 Da) for His-tagged C-terminal domain of Stt3p.

Figure 3

2D NMR [¹H, ¹⁵N] HSQC spectra of the purified [U-¹⁵N] His-tagged C-terminal domain of Stt3p at concentration of ∼0.2 mM in 20 mM phosphate buffer containing 5% D₂O, pH 6.5 in different detergent micelles. The concentrations of detergents were as follows: (A) 1.5% Digitonin, (B) 1% DDM, (C) 300 mM DPC, and (D) 150 mM OG.

Figure 4

2D NMR [¹H, ¹⁵N] HSQC spectrum of the purified [U-¹⁵N] His-tagged C-terminal domain of Stt3p at concentration of ∼0.2 mM in 20 mM phosphate buffer containing 5% D₂O, pH 6.5 containing 100 mM SDS.

Figure 5

2D NMR [¹H, ¹⁵N] HSQC spectra of the purified [U-¹⁵N] His-tagged C-terminal domain of Stt3p (in 20 mM phosphate buffer, 5% D₂O, pH 6.5) as a function of SDS concentration. The inner figure is close-up view of the tryptophan indole amide proton region from the same spectrum. The concentrations of SDS were as follows: (A) 50 mM SDS; (B) 100 mM SDS; (C) 200 mM SDS and (D) 400 mM SDS.

Figure 6

Circular dichroism (CD) spectroscopic analysis of the C-terminal domain of Stt3p at room temperature. (A) far-UV CD spectra of the C-terminal domain of Stt3p in 300 mM DPC and 100 mM SDS detergent micelles. The protein concentration was 10 μM in 20 mM phosphate buffer, pH 6.5. The characteristic double minima at 208 and 222 nm are indicative of significant α-helical content. (B) near-UV CD spectra of the C-terminal domain of Stt3p in 300 mM DPC and 100 mM SDS detergent micelles. The protein concentration was 89 μM, and the buffer conditions were same as for A.

Figure 7

CD spectra of the wild-type and D518E mutant of the C-terminal domain of Stt3p. The data were collected under the same conditions. (A) far-UV CD spectra. The protein concentrations were 10 μM in 20 mM phosphate buffer, pH 6.5, 100 mM SDS. (B) near-UV CD spectra. The protein concentrations were 89 μM in 20 mM phosphate buffer, pH 6.5, 100 mM SDS. (C) intrinsic tryptophan fluorescence spectra. The protein concentrations were 1 μM in 10 mM phosphate buffer, pH 6.5, 100 mM SDS. The introduction of the mutation leads to an intensity quench and blue shift of the spectrum.

Figure 7

CD spectra of the wild-type and D518E mutant of the C-terminal domain of Stt3p. The data were collected under the same conditions. (A) far-UV CD spectra. The protein concentrations were 10 μM in 20 mM phosphate buffer, pH 6.5, 100 mM SDS. (B) near-UV CD spectra. The protein concentrations were 89 μM in 20 mM phosphate buffer, pH 6.5, 100 mM SDS. (C) intrinsic tryptophan fluorescence spectra. The protein concentrations were 1 μM in 10 mM phosphate buffer, pH 6.5, 100 mM SDS. The introduction of the mutation leads to an intensity quench and blue shift of the spectrum.

Figure 8

The impact of the D518E mutation on 2D [¹H, ¹⁵N] -HSQC spectrum. The black spectrum represents the wild-type, while the superimposed red spectrum is of the D518E mutant of the C-terminal domain of Stt3p.

Figure 9

(A) 1D NMR spectrum of methyl-protonated {Ile(δ₁ only), Leu(¹³CH₃, ¹²CD₃), Val(¹³CH₃, ¹²CD₃)} U-{¹⁵N, ¹³C, ²H} labeled sample of the C-terminal domain of Stt3p (30 μM) in phosphate buffer (20 mM, pH 6.5) and 100 mM deuterated SDS. (B) 1D NMR spectrum of 300 μM peptide ligand, Tyr-Asn-Ser-Thr-Ser-Cys-Am, in phosphate buffer (20 mM, pH 6.5) and 100 mM deuterated SDS. (C) STD NMR spectrum of the complex of methyl-protonated {Ile (δ₁ only), Leu(¹³CH₃, ¹²CD₃), Val(¹³CH₃, ¹²CD₃)} U-{¹⁵N, ¹³C, ²H} labeled sample of the C-terminal domain of Stt3p and acceptor peptide substrate (Tyr-Asn-Ser-Thr-Ser-Cys-Am) in phosphate buffer (20 mM, pH 6.5) and 100 mM deuterated SDS. The protein and peptide concentrations are 30 μM and 300 μM, respectively. The spectrum was recorded with a T_1ρ filter, consisting of a 50-ms spin-lock pulse, to eliminate the resonances of the protein. The appearance of peaks a, b, c, d, e and f in STD spectrum, which correspond to the peaks 1, 2, 3, 4, 5 and 6 in the NMR spectrum of acceptor peptide, reveals the C-terminal domain of Stt3p binds to the acceptor substrate of OT.

Figure 10

(A) An expanded region of the overlay of 2D [¹H, ¹⁵N]-HSQC spectra of the [U-¹⁵N]-labeled C-terminal domain of Stt3p (170 μM) showing changes in the chemical shift positions upon addition of increasing concentration of the substrate peptide. Ratios of protein to peptide are: 1:0 (black), 1:0.5 (red), 1:1 (green), 1:5 (blue), 1:10 (yellow), 1:20 (purple), 1:35 (cyan), 1:50 (black), 1:75 (red) and 1:100 (green). (B) The chemical shift perturbation average of four representative resonances are plotted as a function of the concentration of the substrate peptide and fitted using Hill model of Origin^® 7.0 software.

Figure 10

(A) An expanded region of the overlay of 2D [¹H, ¹⁵N]-HSQC spectra of the [U-¹⁵N]-labeled C-terminal domain of Stt3p (170 μM) showing changes in the chemical shift positions upon addition of increasing concentration of the substrate peptide. Ratios of protein to peptide are: 1:0 (black), 1:0.5 (red), 1:1 (green), 1:5 (blue), 1:10 (yellow), 1:20 (purple), 1:35 (cyan), 1:50 (black), 1:75 (red) and 1:100 (green). (B) The chemical shift perturbation average of four representative resonances are plotted as a function of the concentration of the substrate peptide and fitted using Hill model of Origin^® 7.0 software.