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. 1998 May;7(5):1245–1249. doi: 10.1002/pro.5560070520

Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2.

G F Gao 1, U C Gerth 1, J R Wyer 1, B E Willcox 1, C A O'Callaghan 1, Z Zhang 1, E Y Jones 1, J I Bell 1, B K Jakobsen 1
PMCID: PMC2143999  PMID: 9605330

Abstract

A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA-A2 heavy chain, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit.

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Selected References

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  1. Eilat D., Kikuchi G. E., Coligan J. E., Shevach E. M. Secretion of a soluble, chimeric gamma delta T-cell receptor-immunoglobulin heterodimer. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6871–6875. doi: 10.1073/pnas.89.15.6871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Fung-Leung W. P., Schilham M. W., Rahemtulla A., Kündig T. M., Vollenweider M., Potter J., van Ewijk W., Mak T. W. CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell. 1991 May 3;65(3):443–449. doi: 10.1016/0092-8674(91)90462-8. [DOI] [PubMed] [Google Scholar]
  3. Fung-Leung W. P., Wallace V. A., Gray D., Sha W. C., Pircher H., Teh H. S., Loh D. Y., Mak T. W. CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny. Eur J Immunol. 1993 Jan;23(1):212–216. doi: 10.1002/eji.1830230133. [DOI] [PubMed] [Google Scholar]
  4. Gao G. F., Tormo J., Gerth U. C., Wyer J. R., McMichael A. J., Stuart D. I., Bell J. I., Jones E. Y., Jakobsen B. K. Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2. Nature. 1997 Jun 5;387(6633):630–634. doi: 10.1038/42523. [DOI] [PubMed] [Google Scholar]
  5. Garboczi D. N., Hung D. T., Wiley D. C. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3429–3433. doi: 10.1073/pnas.89.8.3429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Garboczi D. N., Utz U., Ghosh P., Seth A., Kim J., VanTienhoven E. A., Biddison W. E., Wiley D. C. Assembly, specific binding, and crystallization of a human TCR-alphabeta with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2. J Immunol. 1996 Dec 15;157(12):5403–5410. [PubMed] [Google Scholar]
  7. Garcia K. C., Degano M., Stanfield R. L., Brunmark A., Jackson M. R., Peterson P. A., Teyton L., Wilson I. A. An alphabeta T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex. Science. 1996 Oct 11;274(5285):209–219. doi: 10.1126/science.274.5285.209. [DOI] [PubMed] [Google Scholar]
  8. Garcia K. C., Scott C. A., Brunmark A., Carbone F. R., Peterson P. A., Wilson I. A., Teyton L. CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes. Nature. 1996 Dec 12;384(6609):577–581. doi: 10.1038/384577a0. [DOI] [PubMed] [Google Scholar]
  9. Giblin P. A., Leahy D. J., Mennone J., Kavathas P. B. The role of charge and multiple faces of the CD8 alpha/alpha homodimer in binding to major histocompatibility complex class I molecules: support for a bivalent model. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1716–1720. doi: 10.1073/pnas.91.5.1716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grégoire C., Lin S. Y., Mazza G., Rebai N., Luescher I. F., Malissen B. Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7184–7189. doi: 10.1073/pnas.93.14.7184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hilyard K. L., Reyburn H., Chung S., Bell J. I., Strominger J. L. Binding of soluble natural ligands to a soluble human T-cell receptor fragment produced in Escherichia coli. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9057–9061. doi: 10.1073/pnas.91.19.9057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Killeen N., Moriarty A., Teh H. S., Littman D. R. Requirement for CD8-major histocompatibility complex class I interaction in positive and negative selection of developing T cells. J Exp Med. 1992 Jul 1;176(1):89–97. doi: 10.1084/jem.176.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Leahy D. J., Axel R., Hendrickson W. A. Crystal structure of a soluble form of the human T cell coreceptor CD8 at 2.6 A resolution. Cell. 1992 Mar 20;68(6):1145–1162. doi: 10.1016/0092-8674(92)90085-q. [DOI] [PubMed] [Google Scholar]
  14. Maddon P. J., Littman D. R., Godfrey M., Maddon D. E., Chess L., Axel R. The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family. Cell. 1985 Aug;42(1):93–104. doi: 10.1016/s0092-8674(85)80105-7. [DOI] [PubMed] [Google Scholar]
  15. Makrides S. C. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev. 1996 Sep;60(3):512–538. doi: 10.1128/mr.60.3.512-538.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Norment A. M., Littman D. R. A second subunit of CD8 is expressed in human T cells. EMBO J. 1988 Nov;7(11):3433–3439. doi: 10.1002/j.1460-2075.1988.tb03217.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Norment A. M., Salter R. D., Parham P., Engelhard V. H., Littman D. R. Cell-cell adhesion mediated by CD8 and MHC class I molecules. Nature. 1988 Nov 3;336(6194):79–81. doi: 10.1038/336079a0. [DOI] [PubMed] [Google Scholar]
  18. Potter T. A., Rajan T. V., Dick R. F., 2nd, Bluestone J. A. Substitution at residue 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes. Nature. 1989 Jan 5;337(6202):73–75. doi: 10.1038/337073a0. [DOI] [PubMed] [Google Scholar]
  19. Sun J., Leahy D. J., Kavathas P. B. Interaction between CD8 and major histocompatibility complex (MHC) class I mediated by multiple contact surfaces that include the alpha 2 and alpha 3 domains of MHC class I. J Exp Med. 1995 Nov 1;182(5):1275–1280. doi: 10.1084/jem.182.5.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Teh H. S., Garvin A. M., Forbush K. A., Carlow D. A., Davis C. B., Littman D. R., Perlmutter R. M. Participation of CD4 coreceptor molecules in T-cell repertoire selection. Nature. 1991 Jan 17;349(6306):241–243. doi: 10.1038/349241a0. [DOI] [PubMed] [Google Scholar]
  21. Zamoyska R. The CD8 coreceptor revisited: one chain good, two chains better. Immunity. 1994 Jul;1(4):243–246. doi: 10.1016/1074-7613(94)90075-2. [DOI] [PubMed] [Google Scholar]
  22. de Smit M. H., van Duin J. Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7668–7672. doi: 10.1073/pnas.87.19.7668. [DOI] [PMC free article] [PubMed] [Google Scholar]

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