Mutation of Cys-67 alters the thermodynamic stability of the human leukocyte antigen HLA0-B*2705.
The B pocket of the class I major histocompatibility complex-encoded protein HLA-B*2705 has recently been suggested to be responsible for the misfolding of this HLA haplotype and thus to induce susceptibility to autoimmune inflammatory diseases. Four mutants of the B*2705 heavy chain were refolded in the presence of three control peptides. The monitoring of the thermal unfolding of the B*2705-peptide complexes by circular dichroism spectroscopy showed that all heterotrimeric mutants were markedly less stable than the corresponding complexes with the wild-type heavy chain. Among the four heavy chain mutations, the C67S change was investigated for unfolding and peptide binding properties because this position may mediate disulfide pair bridging and alter T-cell recognition of HLA-B*2705. Wild-type heterotrimers completely unfold in a single transition at mild acidic pH whereas increase of the pH to mild basic conditions induce only a partial biphasic unfolding. Cys-67 seems to play a crucial role in controlling the thermodynamic stability of the B*2705-peptide complexes as the C67S mutant unfolds faster and with a single transition, independent of pH. Fluorescence polarization and size exclusion chromatography of unfolding intermediates suggest that the peculiar unfolding of the B*2705 wild-type heavy chain cannot be explained by modified peptide binding properties but more likely by the formation of high molecular weight species.
Reinelt
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S
Department of Applied BioSciences, Swiss Federal Institute of Technology, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Dedier
S
S
Asuni
G
G
Folkers
G
G
Rognan
D
D
D003545:Cysteine; D015796:HLA-B27 Antigen / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007962:Leukocytes / Q000276:immunology; D017354:Point Mutation; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D015796:HLA-B27 Antigen; D003545:Cysteine