crystallization papers\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)

Journal logoBIOLOGICAL
CRYSTALLOGRAPHY
ISSN: 1399-0047

Complex assembly, crystallization and preliminary X-ray crystallographic studies of MHC H-2Kd complexed with an HBV-core nonapeptide

aInstitute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100080, People's Republic of China, bGraduate School, Chinese Academy of Sciences (CAS), Beijing 100080, People's Republic of China, cLaboratory of Structural Biology, Tsinghua University, Beijing 100084, People's Republic of China, and dNuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU, England
*Correspondence e-mail: raozh@xtal.tsinghua.edu.cn, george.gao@ndm.ox.ac.uk

(Received 21 April 2004; accepted 5 June 2004)

In order to establish a system for structural studies of the murine class I major histocompatibility antigen complex (MHC) H-2Kd, a bacterial expression system and in vitro refolding preparation of the complex of H-2Kd with human β2m and the immunodominant peptide SYVNTNMGL from hepatitis B virus (HBV) core-protein residues 87–95 was employed. The complex (45 kDa) was crystallized; the crystals belong to space group P2221, with unit-cell parameters a = 89.082, b = 110.398, c = 47.015 Å, α = β = γ = 90°. The crystals contain one complex per asymmetric unit and diffract X-­rays to at least 2.06 Å resolution. The structure has been solved by molecular replacement and is the first crystal structure of a peptide–H-2Kd complex.

1. Introduction

Major histocompatibility complex (MHC) class I molecules (MHC-I) are plasma-membrane proteins that are expressed by virtually all mammalian cells and play a central role in cellular immune recognition. They present short segments (peptides) of intracellularly processed proteins (forming a peptide–MHC-I complex, abbreviated pMHC-I) to the T-cell receptors (TCR) of cytotoxic T lymphocytes (CTL). This type of specific selective recognition triggers T-cell activation through TCR signal transduction, leading to the CTL cell killing of infected cells (Zinkernagel & Doherty, 1974[Zinkernagel, R. M. & Doherty, P. C. (1974). Nature (London), 248, 701-702.]; Haskins et al., 1984[Haskins, K., Kappler, J. & Marrack, P. (1984). Annu. Rev. Immunol. 2, 51-66.]; Townsend et al., 1986[Townsend, A. R., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. & McMichael, A. J. (1986). Cell, 44, 959-968.]; Bjorkman & Parham, 1990[Bjorkman, P. J. & Parham, P. (1990). Annu. Rev. Biochem. 59, 253-288.]), thus conferring cellular immunity against viral infection.

pMHC-I molecules are heterotrimeric structures with (i) a polymorphic membrane-anchored heavy chain with extracellular domains α1, α2 and α3, (ii) a light invariant soluble noncovalently attached β2-microglobulin (β2m) unit and (iii) an 8–11-amino-acid peptide positioned in a cleft formed by the α1 and α2 domains of the heavy chain (Madden, 1995[Madden, D. R. (1995). Annu. Rev. Immunol. 13, 587-622.]).

MHC class I genes are characterized by their extraordinary polymorphism, being the most polymorphic genes known to date, which imparts unique spatial and chemical characteristics to each cleft (Trowsdale & Campbell, 1992[Trowsdale, J. & Campbell, R. D. (1992). Eur. J. Immunogenet. 19, 45-55.]) and in turn dictates the T-cell epitopes (the binding peptides) of each MHC class I allele. Human HLA-A2 (A*0201) was the first crystal structure of an MHC complex to be determined (Bjorkman et al. 1987a[Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L. & Wiley, D. C. (1987a). Nature (London), 329, 506-512.],b[Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L. & Wiley, D. C. (1987b). Nature (London), 329, 512-518.]) and was soon followed by the structures of murine and human MHC molecules complexed with single peptides (reviewed in Madden, 1995[Madden, D. R. (1995). Annu. Rev. Immunol. 13, 587-622.]; Jones, 1997[Jones, E. Y. (1997). Curr. Opin. Immunol. 9, 75-79.]). Analysis of these structures improved our comprehension of how cleft architecture affects both peptide presentation and the conformation of the side chains situated on the α1 and α2 helices bordering the binding cleft.

The purpose of the present study was to establish a system for structural studies of the murine MHC class I H-2Kd. The complex includes an immunodominant peptide derived from hepatitis B virus (HBV) core protein (amino acids 87–96). The structural analysis of the complex H-2Kd–HBV core 87–96 complex is important for several reasons. Firstly, the structure of H-2Kd is surprisingly unknown, although H-2kd-restricted peptide motifs, with the anchor being Tyr at position 2 and Ile or Leu at position 9, have been proposed in previous studies (Falk et al., 1991[Falk, K., Rotzschke, O., Stevanovic, S., Jung, G. & Rammensee, H. G. (1991). Nature (London), 351, 290-296.]; Maryanski et al., 1993[Maryanski, J. L., Luthy, R., Romero, P., Healy, F., Drouet, C., Casanova, J. L., Jaulin, C., Kourilsky, P. & Corradin, G. (1993). Semin. Immunol. 5, 95-104.]). The H-2Kd molecule has been widely used in functional analysis of T-cell recognition and its epitopes include not only many foreign antigens but also autologous antigens (Amrani et al., 2000[Amrani, A., Verdaguer, J., Serra, P., Tafuro, S., Tan, R. & Santamaria, P. (2000). Nature (London), 406, 739-742.]; Fan et al., 2000[Fan, R., Tykodi, S. S. & Braciale, T. J. (2000). J. Immunol. 164, 1669-1680.]); therefore, analysis of the H-2Kd molecular structure will help in understanding the detail of antigen presentation in cellular immunity and the mechanism of autoimmune disorder. Secondly, HBV is a non-cytopathic DNA virus that chronically infects 350 million people worldwide. Like many other chronic viral diseases and cancers, it is associated with T-cell hyper-responsiveness or tolerance (Chisari, 1995[Chisari, F. V. (1995). Hepatology, 22, 1316-1325.]; Chisari & Ferrari, 1995[Chisari, F. V. & Ferrari, C. (1995). Springer Semin. Immunopathol. 17, 261-281.]). HBV transgenic mice have already become a model system for the evaluation of immunotherapeutic strategies to break tolerance and terminate persistent HBV infection (Chisari, 1995[Chisari, F. V. (1995). Hepatology, 22, 1316-1325.]). Mutation within immunodominant CTL epitopes is closely connected with immunological tolerance (McMichael, 1993[McMichael, A. (1993). Science, 260, 1771-1772.]; Bertoletti et al., 1994[Bertoletti, A., Sette, A., Chisari, F. V., Penna, A., Levrero, M., De Carli, M., Fiaccadori, F. & Ferrari, C. (1994). Nature (London), 369, 407-410.]). Therefore, structural knowledge of the complex of H-­2Kd with HBV core 87–96, which is an immunodominant epitope of HBV major antigen, will be of benefit to research on the structural mechanism of immunological tolerance. Thirdly, we have previously found that a 7-mer peptide (YVNTNMG) of the HBV-core antigen (HBcAg 88–94) is associated with heat-shock protein (HSP) gp96 in liver tissues of patients with HBV-induced hepatocellular carcinoma (HCC; Meng et al., 2001[Meng, S. D., Gao, T., Gao, G. F. & Tien, P. (2001). Lancet, 357, 528-529.], 2002). This peptide is highly homologous to mouse H-2Kd-restricted 9-mer peptide (SYVNTNMGL; core 87–95). We have also found that this 7-mer binds to H-­2Kd in vitro (unpublished data), though with a lower affinity than the 9-mer peptide.

We have employed a bacterial expression system and in vitro complex assembly to prepare crystals of H-2Kd with the peptide SYVNTNMGL from HBV core-protein residues 87–95. This H-2Kd-restricted peptide has been shown to elicit CTL responses in H-2d mice with DNA vaccination (Kuhrober et al., 1997[Kuhrober, A., Wild, J., Pudollek, H. P., Chisari, F. V. & Reimann, J. (1997). Int. Immunol. 9, 1203-1212.]). We report here the conditions for successful refolding, purification and crystallization of the complex of H-­2Kd with this HBV-core 87–95 epitope. The crystals diffract X-rays to beyond 2.06 Å and the structure has been solved by molecular replacement.

2. Materials and methods

2.1. Preparation of H-2Kd and β2m proteins as inclusion bodies

To construct the expression vector of H-­2Kd heavy chain and β2m, the region encoding amino acids 1–280 of H-2Kd heavy chain and amino acids 1–99 of human β2m were amplified by PCR and cloned into pET-­3a. The expression plasmids were verified by sequencing and transformed into BL21(DE3)pLysS (Novagen). Transformed BL21(DE3)pLysS cells were grown at 310 K in Luria–Bertani medium containing 50 µg ml−1 carbenicillin. Isopropyl-D-thiogalactopyranoside (IPTG; Sigma) was added to a final concentration of 0.5 mM when the culture reached an OD600 of 0.6.  After a further 3–4 h incubation at 310 K, the bacteria were harvested and suspended in cold phosphate-buffered saline (PBS) buffer. After being lysed using a sonicator and centrifuged at 20 000g, the pellet was washed three times with a solution of 20 mM Tris–HCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 0.5% Triton X-100. H-2Kd and β2m inclusion bodies constituted most of the pellet.

2.2. Preparation of the H-2Kd complex

This was carried out essentially as previously described by Wiley and coworkers (Garboczi et al., 1992[Garboczi, D. N., Hung, D. T. & Wiley, D. C. (1992). Proc. Natl Acad. Sci. USA, 89, 3429-3433.]). Briefly, the H-­2Kd heavy chain and β2m inclusion bodies were separately dissolved in a solution of 10 mM Tris–HCl pH 8.0 and 8 M urea. The synthetically prepared HBV-derived peptide (SYVNTNMGL) was also dissolved in dimethyl sulfoxide (DMSO). H-2Kd heavy chain, β2m and peptide in a 1:1:3 molar ratio were refolded by dilution. After 24–48 h of incubation at 277 K, the soluble portion was concentrated and then purified by chromatography on a Superdex G-75 (Pharmacia) size-exclusion column followed by Mono Q (Pharmacia) anion-exchange chromatography.

2.3. Crystallization of the H-2Kd complex

The purified complex protein (45 kDa) was dialyzed against crystallization buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl) and concentrated to 10 mg ml−1. Initial crystallization conditions were screened using Crystal Screen (Hampton Research). The complex crystallized from conditions containing PEG 20 000. The conditions yielding crystals were further optimized by variation of precipitant and protein concentration and additives. Crystals of good quality can be obtained using 0.1 M MES pH 6.5, 18%(w/v) PEG 20 000, 8%(v/v) DMSO. Crystallization was performed by the hanging-drop vapour-diffusion method at 291 K. 1 µl protein solution was mixed with 1 µl reservoir solution and the mixture was equilibrated against 200 µl reservoir solution at 291 K.

2.4. Data collection and processing

Data collection from the H-2Kd complex was performed in-house on a Rigaku RU-2000 rotating copper-anode X-ray generator operated at 48 kV and 98 mA (Cu Kα; λ = 1.5418 Å) with a MAR 345 image-plate detector. The crystals were mounted in nylon loops and flash-cooled in a cold nitrogen-gas stream at 100 K using an Oxford Cryosystem with reservoir solution as the cryoprotectant. Data were indexed and scaled using DENZO and SCALEPACK (Otwinowski & Minor, 1997[Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307-326.]).

3. Results and discussion

H-2Kd heavy chain could only be refolded in the presence of β2m and peptide (Figs. 1[link]a, 1[link]b and 1[link]c). The refolding resulted in yields of approximately 10% of complex (45 kDa), which could be purified to homogeneity by Superdex G-75 size-exclusion chromatography and Mono Q (Pharmacia) anion-exchange chromatography (Figs. 1[link]a, 1[link]b and 1[link]c). The chromatographic elution profile showed three peaks corresponding to the refolded complex (45 kDa; peak 2), uncomplexed β2m (peak 3) and non-native aggregated products (peak 1; Figs. 1[link]a and 1[link]b). The refolded complex was further purified by Mono Q chromatography and the complex was eluted at an NaCl concentration of 17–26 mM (Fig. 1[link]c). Moreover, we also found that the filtrate of the first refolding solution can be used for further refolding without the addition of peptide if sufficient peptide was added in the first refolding experiment.

[Figure 1]
Figure 1
Purification of the complex of H-2Kd with HBV core 87–96 by FPLC Superdex G75 gel-filtration and Mono Q anion-exchange chromatography. (a) Refolding attempt without peptide. The first peak represents aggregated heavy chain (labelled 1) and the second peak β2m (labelled 3). (b) Refolding in the presence of peptide (SYVNTNMGL). In comparison with the profile in (a), peak 2 represents the correctly refolded H-2Kd complex (45 kDa). (c) Further purification of the refolded complex by anion exchange. Peak 4 represents the H-2Kd complex, which was eluted at a NaCl concentration of 17–26 mM. (d) SDS–PAGE gel (15%) of the purified complex. Lane 1, H-2Kd inclusion bodies; lane 2, β2m inclusion bodies; lane 3, protein standard markers in kDa; lane 4, the purified refolded H-2Kd complex, showing bands for H-2Kd and β2m.

Large single crystals (Fig. 2[link]) appeared in 5 d under optimized conditions. The H-2Kd complex crystals belong to space group P2221, with unit-cell parameters a = 89.082, b = 110.398, c = 47.015 Å, α = β = γ = 90°. Assuming the presence of one molecule in the asymmetric unit, the solvent content is calculated to be about 56%. Selected data statistics are shown in Table 1[link]. Structure determination by molecular replacement using the structure of a murine H-2Db complex (PDB code 1fg2 ; Tissot et al., 2000[Tissot, A. C., Ciatto, C., Mittl, P. R., Grutter, M. G. & Pluckthun, A. (2000). J. Mol. Biol. 302, 873-885.]) as a search model has been successful and the detailed structure will be reported elsewhere.

Table 1
Data-collection and processing statistics of the H-2Kd complex

Space group P2221
Unit-cell parameters (Å) a = 89.082, b = 110.398, c = 47.015, α = 90, β = 90, γ = 90
Wavelength (Å) 1.5418
Redundancy 7.9 (7.7)
Reflection observed 236416
Unique reflections 29503
Completeness (%) 99.9 (100.0)
I/σ(I) 28.5 (8.1)
Rsym (%) 7.9 (37.9)
Rsym = [\textstyle \sum_{h}\sum_{l}|I_{ih} - \langle I_{h}\rangle|/][\textstyle \sum_{h}\sum_{i}\langle I_{h}\rangle], where 〈Ih〉 is the mean of the observations Iih of reflection h.
[Figure 2]
Figure 2
Typical crystals of the H-2Kd–HBV core 87–95 complex, which were used for data collection.

Footnotes

These authors contributed equally to this work.

Acknowledgements

This work was supported by Project 973 of the Ministry of Science and Technology of China (Grant No. 2001CB510001). GFG's stay at the Institute of Microbiology, Chinese Academy of Sciences was supported by a K. C. Wong Fellowship.

References

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Journal logoBIOLOGICAL
CRYSTALLOGRAPHY
ISSN: 1399-0047
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