Peptide binding to the class Ib molecule, Qa-1b.
We have analyzed the interaction of a nonameric peptide (Qdm) derived from the leader sequence of a MHC D region molecule with the class Ib molecule, Qa-1b. Using a direct binding assay with radiolabeled peptide on intact cells, we show specific binding of iodinated Qdm peptide to Qa-1b expressing cells. Specificity was confirmed by experiments in which the binding of iodinated peptide was blocked by unlabeled Qdm and not by control peptides. This allowed us to determine the on- and off-rate of peptide binding to Qa-1b, its binding affinity, and number of available peptide-binding sites per cell. Optimal binding occurs at 4 degrees C, and most of the binding to Qa-1b occurs within the first 10 min and peaks after 3 h. The dissociation of the peptide from cells has a t1/2 of approximately 10 h. The Kd is calculated between 0.2 and 1.1 x 10(-10) M, which is in the range or slightly higher than the Kd we measured for pMCMV to Ld (0.9 x 10(-10) M). The relatively high affinity of binding of the Qdm peptide and low dissociation rate could partially explain why a large portion of Qa-1b molecules are occupied with this single peptide species. We also changed each peptide residue to Ala or Gly to determine its effect on binding. Substitution of Leu at position 9 to Ala strongly reduced peptide binding, while changes at positions 2 (Met-->Ala) and 5 (Arg-->Ala) had a lesser effect. Binding to Qa-1b was completely abrogated with simultaneous Ala mutations at positions 2 and 9.
Kurepa
Z
Z
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Forman
J
J
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000818:Animals; D002460:Cell Line; D000939:Epitopes / Q000378:metabolism; D005998:Glycine / Q000235:genetics; D006183:H-2 Antigens / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D000939:Epitopes; D006183:H-2 Antigens; D015395:Histocompatibility Antigens Class I; D010455:Peptides; C032907:Q surface antigens; C090580:Qdm protein, mouse; C073119:TAP1 protein, human; C486171:Tap1 protein, mouse; D000409:Alanine; D005998:Glycine