Probing the ligand-induced conformational change in HLA-DR1 by selective chemical modification and mass spectrometric mapping.
Peptide binding induces conformational changes in class II MHC proteins that have been characterized using a variety of hydrodynamic and spectroscopic approaches, but these changes have not been clearly localized within the overall class II MHC structure. In this study, empty and peptide-loaded complexes of HLA-DR1, a common class II MHC variant, were chemically modified using the side chain-specific chemical modifiers p-hydroxyphenylglyoxal (arginine), tetranitromethane (tyrosine), N-bromosuccinimide (tryptpophan), and NHS-biotin (lysine). Modified proteins were subjected to in-gel digestion with trypsin and subsequent analysis by MALDI/MS. Three arginine residues and two lysine residues were differentially reactive, modified in the empty form but not the peptide-loaded form of the protein, indicating that the chemical reactivity of these regions differs in the two conformations. Three of the differential modifications were located on a single lateral face of the protein, indicating that this region is involved in the conformational change. Additionally, a number of lysine and tyrosine modification sites were present in both protein conformations. Overall, the pattern of reactivity is inconsistent with the idea that empty MHC molecules exist as molten globules or other partially unfolded intermediates, and suggests that the peptide-induced conformational change is localized to only a few regions of the protein.
Carven
Gregory J
GJ
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Stern
Lawrence J
LJ
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D015798:HLA-DR1 Antigen / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D015798:HLA-DR1 Antigen; D008024:Ligands; D015335:Molecular Probes