pH affects both the mechanism and the specificity of peptide binding to a class II major histocompatibility complex molecule.
We have compared the contribution of electrostatic forces in the binding of antigenic peptides to the class II MHC molecule, IEk, at weakly acidic (pH 5.4) and neutral (pH 7.5) pH values. The binding of specific moth cytochrome c (MCC) and hemoglobin (Hb) peptides to IEk is very sensitive to ionic strength at pH 7.5 but not at pH 5.4, indicating that the mechanism of peptide binding is pH-dependent. Substitution of the C-terminal Lys in MCC for an Ala residue selectively destroyed peptide binding at neutral pH and increased the dissociation rate at least 30-fold, implicating this residue in the pH-dependent electrostatic interaction. The presence of a C-terminal Lys in many of the peptides that are restricted to IEk suggests that this electrostatic interaction is widely used to bind peptides to this MHC molecule. We also probed the electrostatic environment of the peptide binding groove adjacent to the N-terminus of the bound peptide by rapid-diffusion fluorescence energy transfer using a terbium-labeled MCC peptide. In this region of the peptide binding groove, more negative charge is present at pH 7.5 than at pH 5.4. These findings indicate the importance of MHC carboxylates to the mechanism and specificity of peptide binding. The biological importance of having two distinct mechanisms of peptide binding at different pH may be that it acts to broaden the spectrum of antigenic peptides that can be presented to T-cells.
Boniface
J J
JJ
Department of Microbiology and Immunology, Stanford University, California 94305.
Allbritton
N L
NL
Reay
P A
PA
Kantor
R M
RM
Stryer
L
L
Davis
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D010856:Columbidae; D006224:Cricetinae; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014162:Transfection
D003574:Cytochrome c Group; D006454:Hemoglobins; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments