Expression, purification and preliminary X-ray crystallographic analysis of the chicken MHC class I molecule YF1*7.1

Abstract

YF1*7.1 is an allele of a polymorphic major histocompatibility complex (MHC) class I‐like locus within the chicken Y gene complex. With the aim of understanding the possible role of the YF1*7.1 molecule in antigen presentation, the complex of YF1*7.1 heavy chain and β2‐microglobulin was reconstituted and purified without a peptide. Crystals diffracted synchrotron radiation to 1.32 Å resolution and belonged to the monoclinic space group P21. The phase problem was solved by molecular replacement. A detailed examination of the structure may provide insight into the type of ligand that could be bound by the YF1*7.1 molecule.

Brief review of the chicken Major Histocompatibility Complex: the genes, their distribution on chromosome 16, and their contributions to disease resistance

“…At least one MHC-Y class I (YF) molecule is alloimmunogenic (Hunt et al, 2006 ). Initial evidence of MHC-Y class I antigen binding groove specialization (Afanassieff et al, 2001 ; Afanassieff et al 2000 ) was confirmed by structural studies (Figure 2 ) of YF1*7.1 (Hee et al, 2010 ; Hee et al, 2009 ) showing non-peptidic ligands logged within its hydrophobic binding groove. The structure of YF1*7.1 defined a new MHC class I paradigm; a molecule with the structure of a classical MHC class I molecule, but binding “non-classical” non-peptide ligands.…”

Structure of a Classical MHC Class I Molecule That Binds “Non-Classical” Ligands

“…Procedures for protein preparation and crystallization of YF1*7.1 HC and β 2 m have been reported previously [47] . A similar procedure was applied to produce the other YF1*7.1 complexes.…”

“…In the reconstitution experiments, however, the respective lipid was added in 10-fold molar excess to YF1*7.1 HC in a buffer pre-warmed to 37°C, followed by incubation at room temperature for 2 d. All lipids were obtained from Sigma-Aldrich with the following product numbers: palmitic acid (PLM), P5585; oleic acid (OLA), O1008; phosphatidylcholine (PC), P2772; dioleoylphosphatidylcholine (DOPC), P6354; and palmitoyloleoylphosphatidylcholine (POPC), P3017. Lipids were dissolved in dimethylsulfoxide (DMSO) and the concentration adjusted to 10 mg/ml and preheated to ∼40°C prior to adding them to the reconstitution experiments of YF1*7.1 HC and β 2 m. Protein complexes were purified and crystallization experiments were performed as described before [47] . Crystals were cryo-protected with the respective reservoir solution supplemented with either 19% (v/v) glycerol or 19% (v/v) PEG 200.…”

“…X-ray diffraction data sets were collected at BESSY II, Berlin, Germany, at beamline 14.1 or 14.2. Structure solution of the YF1*7.1 complex has been described by us [47] . Molecular replacement for the other structures was performed by employing a search model of ligand- and water-depleted YF1*7.1:L1.…”

HLA class I-associated diseases with a suspected autoimmune etiology: HLA-B27 subtypes as a model system

“…In producing complexes of HLA class I molecules and peptides, we have adapted the protocol described by Garboczi et al (1992) not only to the extracellular HC fragment of HLA-B27 subtypes (Hülsmeyer et al, 2002) and other HLA class I molecules (Table 1) ( [Menssen et al, 1999], [Hillig et al, 2001], [Hülsmeyer et al, 2005a], [Kumar et al, 2009a] and [Kumar et al, 2009b]), but also to MHC class I molecules of the chicken ( [Hee et al, 2009] and [Hee et al, 2010] and unpublished results). For IR spectroscopy, the light chain of MHC class I complexes, β 2 m, was labelled in vivo with 13 C and then complexed with unlabelled HLA-B27 HC and selected peptides in vitro ( [Fabian et al, 2008], [Fabian et al, 2010] and [Fabian et al, 2011]).…”