Preliminary X-ray crystallographic studies of the TRAF domain of human TRAF4

The human TRAF4 TRAF domain was crystallized and the crystals were found to belong to space group P2₁2₁2₁, with unit-cell parameters a = 58.9, b = 87.9, c = 117.3 Å, α = β = γ = 90°. The crystals were obtained at 293 K and diffracted to a resolution of 2.3 Å.

Abstract

TNF receptor-associated factor (TRAF) proteins were initially identified as tumour necrosis factor receptor (TNFR)-interacting proteins that perform critical functions in the regulation of inflammation, antiviral responses and apoptosis. Although TRAF4 is a canonical TRAF protein, it contains a unique domain boundary and functions differently in the cell. In this study, the human TRAF4 TRAF domain, corresponding to amino acids 290–470, was overexpressed in Escherichia coli using engineered C-terminal His tags. The TRAF4 TRAF domain was then purified to homogeneity and crystallized at 293 K. Finally, X-ray diffraction data were collected to a resolution of 2.3 Å from a crystal belonging to space group P2₁2₁2₁, with unit-cell parameters a = 58.9, b = 87.9, c = 117.3 Å, α = β = γ = 90°.

1. Introduction  

TNF receptor-associated factor (TRAF) proteins are key intra­cellular signalling components involved in the tumour necrosis factor receptor (TNFR) and Toll-like receptor (TLR) family signalling pathways that play a critical role in the immune system (Inoue et al., 2000 ▶). TRAFs function as scaffold proteins that link signalling receptors to downstream signalling molecules (Arch et al., 1998 ▶). Seven TRAF proteins, TRAF1–TRAF7, have been identified in mammals (Chung et al., 2002 ▶). Each TRAF exhibits specific biological functions and all TRAF proteins except TRAF7 contain the TRAF domain, which mediates protein interactions (Rothe et al., 1994 ▶).

Even though TRAF4 is a canonical TRAF protein, it contains a unique domain boundary and functions differently in the cell (Kedinger & Rio, 2007 ▶). TRAF4 has a unique nuclear localization signal (NLS) and seven zinc-finger motifs. Although the cellular functions of TRAF4 are not well understood, several studies have indicated that TRAF4 might be important for gross tracheal formation and neural tube formation (Kedinger & Rio, 2007 ▶). The most recent studies have also shown that, like other TRAF family members, TRAF4 plays a role in immune-system function by interacting with NOD-like receptors (Marinis et al., 2011 ▶).

Previously reported crystal structures of the TRAF domains of TRAF2, TRAF3, TRAF5 and TRAF6 have shown that TRAF domains are composed of seven or eight antiparallel β-strand folds followed by a coiled-coil region (Park et al., 1999 ▶; Ye et al., 2002 ▶). TRAF domains usually form mushroom-like trimeric structures in solution (Zhang et al., 2012 ▶). Despite the emerging roles of TRAF4 in human disease states (Izban et al., 2000 ▶; Aston et al., 2004 ▶), including cancer, neuronal diseases and immune diseases, limited structural information is available.

As a first step towards elucidating the molecular structure of TRAF4 and further understanding its molecular interaction with many downstream components, we overexpressed, purified and crystallized the TRAF domain of human TRAF4 corresponding to amino acids 290–470 (hereafter called TRAF4^290–470), after which we purified the domain by affinity chromatography followed by gel-­filtration chromatography. Structural study of protein-interaction modules can often provide critical information on the interaction mode (Bae & Park, 2011 ▶; Park, 2011 ▶). Structural studies of the complex between the TRAF domain and various peptides from binding receptors shed light on TRAF protein-mediated signalling (Ye et al., 2002 ▶). The final crystals of TRAF4^290–470 diffracted to a resolution of 2.3 Å and refinement is in progress. Details regarding the structure of TRAF4^290–470 should help us to better understand TRAF4-mediated signalling events, which are critical to neuronal cell development and immunity.

2. Materials and methods  

2.1. Expression and purification  

To express C-terminally His-tagged protein, human TRAF4^290–470 was amplified by PCR using forward (5′-GGGCATATGCAGGA­GCTGCAGGAGCTT-3′) and reverse (5′-GGGCTCGAGAACAG­CAGCACGGATGAAG-3′) primers. The PCR product was then digested with the NdeI and XhoI restriction enzymes (Enzynomics, Republic of Korea), after which it was inserted into pET24a vector which had been cut with the same restriction enzymes. The plasmid was then transformed into Escherichia coli BL21 (DE3) competent cells, after which its expression was induced by treating the bacteria with 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) overnight at 294 K. 2 l Luria–Bertani (LB) medium was used in this study.

Cells expressing TRAF4^290–470 were pelleted by centrifugation, resuspended and lysed by sonication in 25 ml lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 25 mM imidazole). The lysate was then centrifuged at 16 000 rev min⁻¹ for 30 min at 277 K, after which the supernatant fractions were applied onto a gravity-flow column (Bio-Rad) packed with Ni–NTA affinity resin (Qiagen). The nonspecifically bound bacterial proteins were subsequently removed from the column using wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 60 mM imidazole). The C-terminally His-tagged TRAF4^290–470 was eluted from the column using elution buffer (20 mM Tris buffer pH 8.0, 500 mM NaCl, 250 mM imidazole). The elution fractions were collected on a 0.5 ml scale to 2 ml. The eluted protein was then applied onto a Superdex 200 gel-filtration column (GE Healthcare) that had been pre-equilibrated with a solution consisting of 20 mM Tris pH 8.0, 150 mM NaCl. A gel-filtration standard (Bio-Rad) containing a mixture of molecular-mass markers (thyroglobulin, 670 000 Da; globulin, 158 000 Da; ovalbumin, 44 000 Da; myoglobulin, 17 000 Da; vitamin B₁₂, 1350 Da) was used for size calibration. TRAF4^290–470-containing fractions were collected and concentrated to 9–10 mg ml⁻¹. The peak was then confirmed to contain TRAF4 by SDS–PAGE. Purified TRAF4^290–470 retains the extra C-terminal residues LEHHHHHH.

2.2. Crystallization  

The crystallization conditions were initially screened at 293 K by the hanging-drop vapour-diffusion method using screening kits from Hampton Research (Crystal Screen, Crystal Screen 2, Natrix, MembFac, Index, Crystal Screen Cryo and Crystal Screen Lite) and from Emerald BioSystems (Wizard I, II, III and IV). Initial crystals were grown on the plates by equilibrating a mixture consisting of 1 µl protein solution (9–10 mg ml⁻¹ protein in 20 mM Tris–HCl pH 8.0, 150 mM NaCl) and 1 µl reservoir solution (condition No. 92 from the Index screen: 0.1 M magnesium formate dihydrate, 15% polyethylene glycol 3350) against 0.4 ml reservoir solution. Crystallization was further optimized by searching over a range of concentrations of polyethylene glycol 3350 and magnesium formate dihydrate. Crystals appeared within 3 d and grew to maximum dimensions of 0.5 × 0.2 × 0.1  mm (Fig. 1 ▶) in the presence of 0.14 M magnesium formate dihydrate and 13% polyethylene glycol 3350.

Figure 1.

Crystals of the TRAF domain of human TRAF4. Crystals were grown in 2 d in the presence of 0.14 M magnesium formate dihydrate, 13% polyethylene glycol 3350. The approximate dimensions of the crystals were 0.5 × 0.2 × 0.1 mm.

2.3. Crystallographic data collection  

For data collection at 110 K, the crystals were transiently soaked in a solution corresponding to the reservoir solution supplemented with 30%(v/v) glycerol. The soaked crystals were then flash-cooled in liquid nitrogen. A 2.3 Å resolution native diffraction data set was collected on beamline BL-4A of the Pohang Accelerator Laboratory (PAL), Republic of Korea (Fig. 2 ▶). A total of 180 images were collected with 1° oscillation. The data set was indexed and processed using HKL-2000 (Otwinowski & Minor, 1997 ▶). The data-collection statistics are summarized in Table 1 ▶.

Figure 2.

Diffraction image (1° oscillation) of a crystal of the TRAF domain of TRAF4 with a 2.3 Å resolution limit.

Table 1. Diffraction data for crystals of the TRAF domain of TRAF4.

Values in parentheses are for the outermost resolution shell.

X-ray source	BL-4A, PAL
Wavelength (Å)	0.97950
Space group	P212121
Unit-cell parameters (Å)	a = 58.9, b = 87.9, c = 117.3
Resolution limits (Å)	50–2.3 (2.34–2.30)
No. of observations	199222
No. of unique reflections	27677
Mean I/σ(I)	43.0 (4.2)
Completeness (%)	99.8 (100)
R merge † (%)	8.0 (56.9)

^† R _merge = , where I_i(hkl) is the ith observation of reflection hkl and 〈I(hkl)〉 is the weighted average intensity of all i observations of reflection hkl.

3. Results and discussion  

TRAF4 is considered to be unique among the TRAFs owing to its differences in domain organization and function. The molecular basis of the interactions leading to signalling pathways mediated by TRAF4 is relatively unknown. To understand the TRAF4-mediated signalling pathways, we overexpressed, purified and crystallized TRAF4^290–470, which is responsible for interaction with various downstream signalling components (Marinis et al., 2011 ▶).

TRAF4^290–470 was purified by two chromatographic steps, His-tag affinity chromatography and gel-filtration chromatography, which produced 98% pure target protein. After the affinity-chromatography step, the target protein was concentrated and applied to gel-filtration chromatography. TRAF4^290–470 eluted at approximately 15 ml on gel-­filtration chromatography, which corresponds to a molecular mass of 70 000 Da. Since the calculated monomeric molecular mass of TRAF4^290–470 including the C-terminal His tag was 23 125 Da, TRAF4^290–470 appears to exist as a trimer in solution, similar to TRAF2 and TRAF5.

We initially tried to obtain a crystal of the target protein using the TRAF-C domain consisting of amino acids 310–470. This was easily crystallized, but never diffracted to better than 6 Å resolution. The success in obtaining a highly diffractable crystal of TRAF4^290–470 was the result of producing a longer construct that contained an extra coiled coil. The domain construct containing amino acids 290–470 diffracted to 2.3 Å resolution. The crystals belonged to space group P2₁2₁2₁, with unit-cell parameters a = 58.9, b = 87.9, c = 117.3 Å, α = β = γ = 90°. Assuming the presence of one trimer in the crystallographic asymmetric unit, the Matthews coefficient (V _M) was calculated to be 2.22 ų Da⁻¹, which corresponds to a solvent content of 44.64% (Matthews, 1968 ▶). Diffraction data statistics are given in Table 1 ▶. The molecular-replacement phasing method was conducted with Phaser (McCoy, 2007 ▶) using the structure of TRAF5 (PDB entry 4gjh; Zhang et al., 2012 ▶), which has 33% amino-acid sequence homology to the TRAF4 TRAF domain, as a search model. Probable solutions with rotation-function and translation-function Z-­scores of 5.2 and 8.1, respectively, for molecule A, of 4.4 and 13.4, respectively, for molecule B, and of 4.0 and 16.0, respectively, for molecule C were initially obtained. Initial refinement with REFMAC5 (Murshudov et al., 2011 ▶) using the initial Phaser model gave an R _work of 34.8% and an R _free of 39.6%. There was one trimer in the asymmetric unit. Further structural refinement is currently being conducted.