Backbone ¹H, ¹⁵N, and ¹³C resonance assignments for the NOXO1β PX domain

Abstract

NOXO1 (Nox Organizer 1) is a homolog of the NAPDH oxidase protein p47^phox. NADPH oxidases transfer electrons from NADPH to molecular oxygen, generating the superoxide anion. NOXO1 contains an N-terminal PX (phox homology) domain and is one of several PX domain-containing proteins found in the cytosolic subunits of the NADPH oxidase complex. These PX domains bind to membrane lipids and target the protein to membranes, recruiting other cytosolic components to the membrane bound components and aiding formation of a active enzyme complex. This recruitment represents a level of regulation of these oxidases. Here we report the backbone assignments of NOXO1β PX.

Biological context

The NADPH oxidases are multiprotein enzyme complexes that generate reactive oxygen species (ROS). ROS generated by these complexes are involved in numerous biological processes including host immune response, cell growth and differentiation, signaling and cell death (reviewed in Krause and Bedard 2007). These complexes include both membrane-bound and cytosolic components. Active complex requires assembly of the cytosolic components with the membrane-bound components at the plasma membrane.

Several of the cytosolic proteins in the NADPH complexes contain a Phox homology (PX) domain. PX domains bind to phosphorylated lipids, most notably the phosphatidylinositol phosphates (Sato et al. 2001). One of these PX domain-containing cytosolic proteins is the NADPH oxidase organizer protein NOXO1. NOXO1 is a 376 residue protein that acts as an organizer within the NADPH oxidase complex to recruite the activator protein, NOXA1, to the membrane-bound catalytic subunit (Banfi et al. 2003, Geiszt et al. 2003 and Takeya et al. 2003). This colocalization represents a major regulatory element of ROS generation. One additional feature of NOXO1 is the presence of four splice variants termed α, β, γ and δ, with NOXO1β being the predominant isoform (Cheng and Lambeth 2005). These splice variants result in insertions and/or deletions within the PX domain. Here, we report the backbone ¹H, ¹³C and ¹⁵N assignments for the NOXO1β PX domain (residues 1–144) and discuss additional methods used to obtain the chemical shift assignments.

Experimental methods

NOXO1β PX Expression and Purification

The sequence for human NOXO1β PX (1–144) was cloned into pGEX-6P-1 (GE Biosciences) and transformed into the E. coli strain BL21(DE3)Codon + RIPL (Stratagene). Cultures were grown at 32°C in minimal media containing 1 g/l ¹⁵NH₄Cl and 2 g/l ¹³C₆ glucose (²H₇¹³C₆ glucose for ²H samples) until OD₆₀₀ reached 0.6 and induced with 0.5 mM IPTG at 22°C overnight (¹H media) or for 72 h (²H media). Stable isotopes were purchased from Isotec/ Sigma and Cambridge Isotope Laboratories. The harvested cell pellet was resuspended in lysis buffer (20 mM Tris, 20 mm NaCl, 2 mM DTT, 0.1% (v/v) Tween-20, pH 8.0) and lysed on ice by sonication (8 cycles, 4 min/cycle, level 8, 30% duty cycle, 1/2″ probe, Branson Sonifier 250). The lysate was incubated with 10 μl Benzonase (EMD) at room temperature for one hour to digest bacterial DNA. Lysates were clarified by centrifugation for 45 min at 30,000 × g at 4°C. Clarified lysate containing GST-NOXO1β PX was run over SP Sepharose resin equilibrated in SP-A buffer (50 mM NaP_i, 100 mM NaCl, 2 mM DTT, pH 7.4). The fusion protein was eluted with a salt gradient (SP-B, 50 mM NaP_i, 1.5 M NaCl, 2 mM DTT, pH 7.4) and eluted at ∼800 mM NaCl. The combination of Benzonase treatment and use of SP Sepharose, rather than GSH Sepharose, for initial capture of the fusion protein eliminated nucleic acid contamination of the product.

The pooled SP Sepharose peak was loaded onto GSH-Sepharose FF resin (GE Biosciences) equilibrated in GSH-A (50 mM NaP_i, 800 mM NaCl, 2 mM DTT, pH 7.4). GST-NOXO1β PX was eluted with 15 mM reduced glutathione in GSH-A. The eluted GSH-Sepharose peak was dialyzed against 2l SP-A overnight at 4°C in the presence of 80 μg PreScission Protease (GE Biosciences) to cleave the GST. The digested fusion protein was loaded onto Source 15S resin (GE Biosciences) equilibrated in SP-A. Cleaved NOXO1β PX was eluted with a salt gradient (SP-B), with the protein eluting at ∼400 mM NaCl. The pooled NOXO1β PX was treated with Pefabloc PSC (0.1 mg/ml final concentration) for 30 min at 4°C. Final yield of purified NOXO1β PX was ∼3 mg/l culture.

Data Collection

NMR spectra were acquired at 293 K on a Bruker Avance DRX-600 spectrometer equipped with a triple resonance Z-gradient cryoprobe. For NMR experiments, NOXO1β PX was dialyzed into 100 mM NaP_i, 100 mM NaCl, 0.1 mM EDTA, pH 6.9. ²H₅ glycerol was added to 5% (v/v) after samples were concentrated. NOXO1β PX was concentrated to ∼500 μM for all NMR experiments. Standard pulse sequences were used to acquire 2D HSQC and 3D HNCA, HNCO, HN(CA)CB, HN(CO)CA, HN(CA)CO and HN(COCA)CB data. NMR data were processed using NMRPipe (Delaglio et al. 1995) and analyzed using NMRView (Johnson et al. 1994).

Assignments and data deposition

Backbone assignments for NOXO1β PX have been made for 125 of 131 non-proline residues (Fig. 1). We have assigned 95% of HN/N, 89% of C^α, 82% of C^β and 90% of C′ atoms, 7/7 Gln sidechain NH₂ resonances 2/2 Trp indole NH resonances. ¹³C chemical shift deviations from consensus CSI values (Wishart and Sykes 1994) and secondary structure predicted by TALOS+ (Shen et al. 2009) are shown in Fig. 2. Predicted secondary structure of NOXO1β PX is consistent with observed secondary structure in other solved PX domains. Backbone assignments for NOXO1β PX have been deposited in the BMRB with accession code 16749.

Fig. 1.

Assigned ¹⁵N-¹H HSQC of NOXO1β PX. The spectrum was obtained with ∼ 500 μM NOXO1β PX in 100 mM NaP_i, 100 mM NaCl, 0.1 mM EDTA, 5% (v/v) ²H₅ glycerol, pH 6.9

Fig. 2.

Δδ (¹³C) for a C^α, b C^β, and c C′ from CSI consensus values, referenced to DSS. d TALOS + predicted secondary structure of NOXO1β PX. Residue number is indicated along the x-axis and the secondary structure is indicated along the y-axis with a value of +1 for β-sheet and −1 for α-helix. The height of the bars indicate the probability of the residue being either a β-sheet (+1 maximum) or α-helix (−1 maximum). Major PX domain structural elements are labeled

Although NOXO1β PX is a small domain (∼16 kDa MW), perdeuteration of the protein was necessary to obtain backbone carbon assignments. With the ¹H/¹⁵N/¹³C-labeled protein, only 152 total C^α peaks and 15 total C^β peaks were observed in HNCA and HNCACB spectra, respectively. In contrast, the ²H/¹⁵N/¹³C NOXO1β PX yielded 253 C^α peaks and 182 C^β peaks in HNCA and HNCACB spectra, sufficient to allow resonance assignment. We hypothesize that the poor magnetization transfer observed in the protonated protein was a result of a longer τ_c due to the addition of glycerol to the sample buffer and the need for running experiments at a cooler temperature (293 K).

While cryogenic probes are able to yield a marked increase in signal-to-noise relative to standard probes, they are sensitive to electrically conductive samples and yield reduced S/N in high-salt buffers. For NOXO1β PX solubility and stability, it was necessary for the sample buffer to contain high levels of both phosphate and salt. Sodium phosphate and sodium chloride were chosen over their potassium counterparts due to a slightly lower conductivity (Kelly et al. 2002). Voehler et al. (2006) reported that a smaller diameter NMR tube is a viable avenue for increasing S/N of high conductivity samples in a cryogenic probe, and we have used a 4 mm Shigemi tube in these experiments in order to decrease requisite ¹H power and to increase S/N. Specifically, ∼ 180 μl of the protein sample was put into a 4 mm Shigemi tube which was placed inside a 5 mm flat bottomed tube containing D₂O. Use of D₂O in the outer tube allowed spectrometer lock without H^N/D^N exchange dilution effects to the protein sample. Although the sample volume within the detect coil decreases by ∼ 30%, the S/N of data acquired in a 4 mm tube is higher than that for a sample of the same concentration in a 5 mm tube. Utilizing a smaller tube also decreased the ¹H 90° pulse from 15 μs to 10.2 μs.