Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the chicken CD8αα–BF2*0401 complex

A crystal of the chicken MHC class I molecule BF2*0401 in complex with chicken CD8αα (CD8αα–BF2*0401) diffracted to 2.8 Å resolution and belonged to space group P2₁, with unit-cell parameters a = 90.6, b = 90.8, c = 94.9 Å, β = 98°.

Abstract

In the process of antigen presentation, the MHCI–CD8 complex is important for immune signal transduction by the activation of cytotoxic T cells. Here, the expression, purification, crystallization and X-ray analysis of the complex of the chicken MHC class I molecule BF2*0401 and CD8αα (CD8αα–BF2*0401) are reported. This complex was verified by SDS–PAGE analysis of a CD8αα–BF2*0401 crystal, which showed three bands corresponding to the molecular weights of BF2*0401, β ₂-microglobulin and CD8α, respectively. The crystal of CD8αα–BF2*0401 diffracted to 2.8 Å resolution and belonged to space group P2₁, with unit-cell parameters a = 90.6, b = 90.8, c = 94.9 Å, β = 98°. The Matthews coefficient and solvent content were calculated to be 2.88 ų Da⁻¹ and ∼57.3%, respectively.

1. Introduction  

During cellular immunity, antigen presentation by MHC class I molecules is the first step in the activation of cytotoxic T cells (CTLs). In this process, only with the engagement of CD8 are MHC class I molecules capable of presenting peptides to T-cell receptors (TCRs; Neefjes et al., 2011 ▶). The CD8αα–MHC class I molecule complex not only enhances the interaction between TCRs and MHCI peptides but also improves their binding sensitivity (Holler & Kranz, 2003 ▶). To date, only three structures of mammalian MHC class I alleles in complex with CD8αα have been determined (Gao et al., 1997 ▶; Kern et al., 1998 ▶; Shi et al., 2011 ▶). All known CD8αα–MHCI structures show that the CD8αα homodimer uses all six complementarity-determining region (CDR)-like loops to bind the CD loop of the MHCI α3 domain in an antibody-like manner. However, the structure of the CD8αα–MHCI complex in non-mammals such as chickens remains unknown.

Chickens are presently being assailed by emerging infectious pathogens such as highly pathogenic avian influenza virus subtype H5N1, which can threaten human health. In chicken immunity, a single dominantly expressed class I allele has been reported to be associated with pathogen resistance (Koch et al., 2007 ▶). The chicken MHC class I molecule BF2*2101 has been reported to confer resistance to Marek’s disease, and its antiviral capacity is related to effective antigen presentation by the MHC and an induced strong CTL response (Wallny et al., 2006 ▶; Garcia-Camacho et al., 2003 ▶). Hence, it is essential for us to have a good understanding of chicken immunity, especially of the CD8αα–MHCI structure-based CTL responses.

In this study, the crystallization and preliminary crystallographic analysis of the CD8αα–BF2*0401 complex are reported for the first time. The results will contribute to understanding the structural characteristics of CD8αα–MHCI in bird species.

2. Materials and methods  

2.1. Macromolecule production  

A DNA fragment encoding the chicken CD8α mature peptide residues 1–118 (GenBank AY528647) was cloned from cDNA synthesized from mRNA extracted from the spleen of a specific pathogen-free (SPF) chicken. The purified CD8α segment was ligated into pET-21a(+) vector (Novagen, Merck KGaA, Darmstadt, Germany) and expressed in Escherichia coli strain BL21 (DE3) (Table 1 ▶). The recombinant protein was expressed as inclusion bodies. The bacteria were harvested and lysed by sonication, and the inclusion bodies were washed three times with washing buffer and dissolved in guanidinium chloride (Gua–HCl) buffer to a concentration of 30 mg ml⁻¹ (Sun et al., 2013 ▶). 3 ml chicken CD8α inclusion bodies was then added gradually to 500 ml refolding buffer (100 mM Tris–HCl pH 8.0, 2 mM EDTA, 400 mM l-arginine–HCl, 0.5 mM oxidized glutathione, 5 mM reduced glutathione) and incubated at 277 K for 8 h. The soluble protein was then concentrated and purified by chromatography on a Superdex 200 16/60 HiLoad (GE Healthcare) size-exclusion column (Zhang et al., 2010 ▶).

Table 1. Chicken CD8αα–BF2*0401 production information.

Source organism	Gallus gallus
DNA source	Cloning from cDNA
Forward primer	ACGCATATGGCCCAGGGCCAGCGTAACACG
Reverse primer	CCGCTCGAGGAAGAAGGCGGGTTGTCCCGAG
Cloning vector	pMD18T
Expression vector	pET-21a(+)
Expression host	E. coli
Complete amino-acid sequence of the construct produced
Chicken CD8α	AQGQRNTMEARFLNRNMKHPQEGQPLELECMPFNIDNGVSWIRQDKDGKLHFIVYISPLSRTAFPRNERTSSQFEGSKQGSSFRLVVKNFRAQDQGTYFCIANINQMLYFSSGQPAFF
BF2*0401	ELHTLRYIRTAMTDPGPGQPWFVTVGYVDGELFVHYNSTARRYVPRTEWIAANTDQQYWDGQTQIGQLNEQINRENLGIRQRRYNQTGGSHTVQWMFGCDILEDGTIRGYRQSAYDGRDFIALDKDMKTFTAAVPEAVPTKRKWEEESEPERWKNYLEETCVEWLRRYVEYGKAELGRRERPEVRVWGKEADGILTLSCRAHGFYPRPIVVSWLKDGAVRGQDAHSGGIVPNGDGTYHTWVTIDAQPGDGDKYQCRVEHASLPQPGLYSW
Chicken β 2m	DLTPKVQVYSRFPASAGTKNVLNCFAAGFHPPKISITLMKDGVPMEGAQYSDMSFNDDWTFQRLVHADFTPSSGSTYACKVEHETLKEPQVYKWDPEF

The pET-21a plasmids of BF2*0401 (GenBank AM282693, residues 1–270 of the mature protein) and chicken β₂-microglobulin (β₂m; GenBank AB178590, residues 1–98 of the mature protein) were kept in our laboratory (Zhang et al., 2012 ▶). Peptide IE8 (IDWFDGKE) was synthesized and purified by HPLC (Scilight-Peptide). The extraction of BF2*0401 and chicken β₂m inclusion bodies and the preparation of refolded BF2*0401 complex were performed essentially as described previously (Sun et al., 2013 ▶; Garboczi et al., 1996 ▶). The peptide IE8 dissolved in dimethyl sulfoxide (DMSO) and the BF2*0401 and β2m inclusion bodies were then refolded in a 3:1:1 molar ratio using the dilution method. After refolding, the pBF2*0401 complex was purified by Superdex 200 16/60 HiLoad (GE Healthcare) and Resource Q anion-exchange (GE Healthcare) chromatography (Zhang et al., 2012 ▶).

2.2. Crystallization of the CD8αα–BF2*0401 complex  

The purified chicken CD8αα and pBF2*0401 were concentrated to 20 mg ml⁻¹ and mixed in a 1:1 molar radio at 277 K overnight. The extinction coefficients of chicken CD8αα and pBF2*0401 were estimated to be 0.73 and 2.36 mg ml⁻¹ at 280 nm, respectively, using ProtParam (http://web.expasy.org/protparam/). The protein concentration was determined using the BCA kit (Novagen, Gemany). The CD8αα–BF2*0401 complex was then concentrated to 5 and 10 mg ml⁻¹ and mixed with reservoir buffer in a 1:1 ratio. Finally, the complex protein was crystallized by the sitting-drop vapour-diffusion method at 291 K. After 15 d, crystals of the chicken CD8αα–BF2*0401 complex were obtained (Table 2 ▶). The complex crystals were picked out from the crystallization drops and washed three times with the reservoir solution for SDS–PAGE analysis.

Table 2. Crystallization conditions.

Method	Sitting-drop vapour diffusion
Plate type	VDX plate
Temperature (K)	291
Protein concentration (mg ml−1)	5, 10
Buffer composition of protein solution	20 mM Tris–HCl pH 8.0, 50 mM NaCl
Composition of reservoir solution	0.2 M ammonium sulfate, 0.1 M Tris pH 8.5, 25%(w/v) polyethylene glycol 3350
Volume and ratio of drop	1:1
Volume of reservoir (µl)	160

2.3. Data collection and processing  

The crystal was first soaked in a cryoprotectant containing 17%(v/v) glycerol for a few seconds and then flash-cooled in liquid nitrogen. Diffraction data were collected on beamline BL17U at the Shanghai Synchrotron Radiation Facility (SSRF; Shanghai, People’s Republic of China) at a wavelength of 0.97972 Å using an ADSC Q315 CCD detector. The collected intensities were indexed, integrated, corrected for absorption, scaled and merged using HKL-2000 (Otwinowski & Minor, 1997 ▶; Table 3 ▶).

Table 3. Data-collection statistics for chicken CD8αα–BF2*0401.

Values in parentheses are for the outer shell.

Diffraction source	BL17U, SSRF
Wavelength (Å)	0.97972
Temperature (K)	100
Detector	ADSC Q315
Crystal-to-detector distance (mm)	300
Rotation range per image (°)	1
Total rotation range (°)	360
Exposure time per image (s)	0.8
Space group	P21
Unit-cell parameters (Å, °)	a = 90.6, b = 90.8, c = 94.9, α = 90.0, β = 98.6, γ = 90.0
Mosaicity (°)	1.0
Resolution range (Å)	50.00–2.80 (2.90–2.80)
Total No. of reflections	163307
No. of unique reflections	37579
Completeness (%)	99.8 (100.0)
Multiplicity	4.3 (4.5)
〈I/σ(I)〉	14.140 (2.630)
R merge (%)†	10.5 (61.3)
Overall B factor from Wilson plot (Å2)	52.44

^† R _merge = , where I_i(hkl) is the observed intensity and 〈I(hkl)〉 is the average intensity from multiple measurements.

3. Results and discussion  

Multiple amino-acid sequence alignments between the chicken proteins and their mammalian counterparts (PDB entries 1akj, 1bqh and 3qzw; Gao et al., 1997 ▶; Kern et al., 1998 ▶; Shi et al., 2011 ▶) were performed using the DNAMAN program. The alignment results showed that the chicken BF2*0401 molecule has 41.7, 42.8 and 42.8% identity to the HLA-A*0201, HLA-A*2402 and H-2K^b molecules, respectively, while the chicken CD8α molecule shares 21.7 and 17.9% sequence identity with human and mouse CD8α, respectively.

After refolding in vitro, approximately 10–12% yields of soluble chicken CD8α and pBF2*0401 proteins were obtained. The soluble chicken CD8α protein was purified by Superdex 200 16/60 HiLoad size-exclusion chromatography (Fig. 1 ▶ a). SDS–PAGE analysis of chicken CD8α showed one clear band at about 13.6 kDa (see inset in Fig. 1 ▶). According to the reference elution profile of the column, this shows that chicken CD8α exists in a homodimeric form (∼28 kDa). The refolded pBF2*0401 complex was also first purified by Superdex 200 16/60 HiLoad size-exclusion chromatography (GE Healthcare; Fig. 1 ▶ b). The refolded protein (peak 2) was further purified by Resource Q anion-exchange chromatography. A single and specific elution peak appeared when the NaCl concentration of the buffer was 10–15% (Fig. 1 ▶ c). SDS–PAGE analysis showed two clear bands corresponding to the expected molecular weights of BF2*0401 (∼32 kDa) and chicken β₂m (∼11 kDa) (see inset in Fig. 1 ▶).

Figure 1.

Purification of the soluble chicken CD8α and pBF2*0401 proteins by Superdex 200 16/60 HiLoad gel-filtration and Resouce Q anion-exchange chromatography (GE Healthcare). (a) The profile peak represents the chicken CD8α homodimer (∼28 kDa). Inset, reduced SDS–PAGE gel (15%) for the peak. Lane M contains molecular-mass markers (labelled in kDa). (b) Gel-filtration profile of the pBF2*0401 complex. Peaks 1, 2 and 3 correspond to the aggregated BF2*0401 heavy chain, the BF2*0401 complex (∼43 kDa) and excess chicken β₂m, respectively. Inset, reduced SDS–PAGE gel (15%) for peaks 1, 2 and 3. Lane M contains molecular-mass markers (labelled in kDa). (c) Anion-exchange chromatography profile of the refolded pBF2*0401 complex, which was eluted at an NaCl concentration of 10–15%. Inset, reduced SDS–PAGE gel (15%) for the peak. Lane M contains molecular-mass markers (labelled in kDa)

On initial crystallization screening, crystals appeared after 15 d (Fig. 2 ▶). SDS–PAGE analysis of the crystals displayed three clear bands corresponding to BF2*0401, CD8α and β₂m (Fig. 3 ▶). In addition, the CD8αα–BF2*0401 complex crystal displayed a good diffraction pattern (Fig. 4 ▶). The Matthews coefficient value V _M was 2.88 ų Da⁻¹, with a calculated solvent content of ∼57.3%.

Figure 2.

Photograph of the crystal used for diffraction analysis.

Figure 3.

SDS–PAGE for identification of the chicken CD8αα–BF2*0401 crystal (lane S). Lane M contains molecular-mass markers (labelled in kDa).

Figure 4.

Diffraction pattern of the chicken CD8αα–BF2*0401 complex; spots corresponding to high-resolution diffraction are highlighted in the box.

Solution of the CD8αα–BF2*0401 complex crystal structure will reveal specific structural features of the CD8αα–MHCI complex in chickens and will contribute to a better understanding of the details of the interaction between CD8αα and MHCI in birds.