Identification of a dominant self‐ligand bound to three HLA B44 alleles and the preliminary crystallographic analysis of recombinant forms of each complex

FEBS Letters

Williams, David S¹  Gorman, Jeffery J³  Macdonald, Whitney¹  Purcell, Anthony W¹  Rossjohn, Jamie²  Clements, Craig S²  Brooks, Andrew G¹  McCluskey, James¹  Kjer-Nielsen, Lars¹ 

[1] Department of Microbiology and Immunology, University of Melbourne, Melbourne, Vic. 3010, Australia;The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biological Sciences, Monash University, Clayton, Vic. 3168, Australia;CSIRO Division of Health Science and Nutrition, 343 Royal Parade, Parkville, Vic. 3052, Australia

关键词: X-ray crystallography;    HLA;    Mass spectrometry;    Protein structure;    Polymorphism;    Peptide ligand;   

DOI  :  10.1016/S0014-5793(02)03149-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A naturally processed and presented ligand that is shared by human leukocyte antigen (HLA) B*4402, B*4403 and B*4405 molecules has been identified in peptides isolated from immunoaffinity purified HLA B44 complexes. This peptide derived from HLA DPα residues 46–54, an endogenous product of HLA DP expressed in the cell line Hmy2.C1R, is a prominent peptide in the mass spectra of species isolated as bound peptides from each allele when the three HLA B44 subtypes were introduced as transfected gene products. Recombinant truncated forms of HLA B*4405_1–276, HLA B*4403_1–276, HLA B*4402_1–276 and β₂-microglobulin have been prepared as inclusion bodies in Escherichia coli and refolded in the presence of the DPα_46–54 peptide and purified by a combination of size exclusion and anion exchange chromatography. This material was determined to be correctly folded based on detection of a conformational epitope recognized by the W6/32 monoclonal antibody. Large, plate-like crystals of the three complexes were produced using polyethylene glycol as the precipitant. All the crystals belong to the space group P2₁2₁2₁ with unit cell dimensions of approximately a=51, b=82, c=110 Å. The crystals of three B44/DPα complexes diffracted to a resolution of 1.9 Å or better. For the first time, using this natural, high abundance ligand of the HLA B44 molecules we have successfully expressed and refolded the three HLA B44 molecules and produced crystals amenable to structural studies.

【 授权许可】

Unknown