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

Abstract

A naturally processed and presented ligand that is shared by human leukocyte antigen (HLA) B*4402, B*4403 and B*4405 molecules has been identi¢ed in peptides isolated from immunoa⁄nity puri¢ed HLA B44 complexes. This peptide derived from HLA DPK K 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 1À276 , HLA B*4403 1À276 , HLA B*4402 1À276 and L L 2 -microglobulin have been prepared as inclusion bodies in Escherichia coli and refolded in the presence of the DPK K 46À54 peptide and puri¢ed 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 P2 1 2 1 2 1 with unit cell dimensions of approximately a = 51, b = 82, c = 110 A î . The crystals of three B44/DPK K complexes di¡racted to a resolution of 1.9 A î or better. For the ¢rst 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. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

High Resolution Structures of Highly Bulged Viral Epitopes Bound to Major Histocompatibility Complex Class I

“…Expression, Purification, and Crystallization of HLA-B35 Alleles in Complex with Long Epitopes-Soluble HLA-B*3501 and HLA-B*3508 molecules (residues 1-276) and full-length ␤ 2 -microglobulin (residues 1-99) were expressed, refolded with the LPEPLPQGQLTAY peptide, purified, and concentrated to 10 mg/ml as described previously (38). The HLA-B35 crystals were obtained by the hanging drop vapor diffusion technique.…”

Impact of clonal competition for peptide-MHC complexes on the CD8+ T-cell repertoire selection in a persistent viral infection

“…25 Crystals of the HLA B*3508-CPS and HLA B*3508-FPT complexes were formed at 4°C with the hanging-drop vapor-diffusion technique, in which an equal volume of HLA B*3508-CPS or HLA B*3508-FPT (10 mg/mL) was mixed with the reservoir (200 mM ammonium acetate, 16% PEG 3350, and 100 mM cacodylate, pH 7.6).…”

CTL Recognition of a Bulged Viral Peptide Involves Biased TCR Selection

“…Protein expression, crystallization, and structure determination Soluble HLA-B*3501 (residues 1-276) and full-length ␤ 2 -microglobulin (residues 1-99) were expressed in Escherichia coli as inclusion bodies, refolded with the EPLPQGQLTAY peptide, and purified as previously described (26,27). The HLA-B*3501 complex crystals were obtained by the hanging drop vapor diffusion technique.…”

“…Since HLA-B*3501 prefers peptide ligands with proline as a dominant anchor residue at position (P) 2, along with a large hydrophobic residue at the C terminus (33-35), it was not possible to predict the binding register of the EPLP 11-mer peptide with certainty since either of the two proline residues at P2 and P4 could serve as potential anchor residues. Moreover, HLA-B*3501 molecules are able to bind the overlapping BZLF1 9-mer ( 56 LPQGQLTAY 64 ) and 11-mer ( 54 EPLPQGQLTAY 64 ) with equal affinity, even though only the longer 11-mer peptide is immunogenic in HLA-B*3501 individuals (27). The crystal structure of the B*3501/11-mer complex was solved to 1.7 Å resolution (R fac , 21.9%; R free , 24.5% ; Table IV) and demonstrated unequivocally that P2-Pro formed a dominant anchor residue of the EPLP peptide (Fig.…”