Abstract
Major Histocompatibility Complex class II (MHC-II) molecules bind peptides and present them to receptors on CD4+ T cells as part of the immune system’s surveillance of pathogens and malignancy. In the absence of peptide, MHC-II equilibrates between peptide-receptive and peptide-averse conformations. The conversion between these forms has been postulated to be important in regulating cellular antigen presentation but has been difficult to study. In order to generate the MHC-II molecule HLA-DR1 in the peptide-receptive form, we designed and tested a series of photocleavable peptides that included the UV-sensitive 3-amino-3-(2-nitrophenyl)-propionate amino acid analog. They were intended to bind tightly to the HLA-DR1 MHC molecule, but to generate low-affinity fragments after UV exposure that would be released to yield HLA-DR1 in the peptide-receptive conformation. We were able to identify photocleavable peptides that bound tightly to HLA-DR1 and generated the peptide-receptive conformation after UV exposure. However, slow release of photocleaved peptide fragments from the binding site limited the rate of binding of an incoming labeled peptide and complicated kinetic measurements of the individual steps of the overall peptide binding reaction. Modification of the N-terminal region of the photocleavable peptide to reduce MHC-II pocket or H-bonding interactions allowed for generation of the peptide receptive form immediately after UV exposure with peptide fragments neither retained within the site nor interfering with binding of an incoming peptide. However this was achieved only at the expense of a substantial reduction in overall peptide binding affinity, and these peptides had such weak interaction with HLA-DR1 that they were easily exchanged by incoming peptide without UV exposure. These results show that photocleavable peptides can be used to generate peptide-receptive HLA-DR1 and to facilitate peptide exchange in generation of specific peptide-MHC-II complexes, but that usage of these peptides for kinetic studies can be constrained by slow fragment release.
Highlights
Major histocompatibility complex (MHC) molecules are membrane glycoproteins that bind short peptides and present them at the cell surface for interaction with receptors on T cells
In order to design a photocleavable peptide for DR1, we used the photolabile amino acid analog 3-amino-3-(2-nitrophenyl)-propionyl residue (Fig 1A) that under UV exposure rearranges so that an oxygen attacks the α-carbon cleaving the peptide backbone
Other important MHC class II proteins (MHC II)-peptide interactions include hydrogen bonds between the peptide backbone and MHC II alpha and beta chain, which are distributed along the entire peptide length [21,34,35,36,37]
Summary
Major histocompatibility complex (MHC) molecules are membrane glycoproteins that bind short peptides and present them at the cell surface for interaction with receptors on T cells This is part of the antigen presentation mechanism by which the immune system recognizes and clears pathogens and tumors. Even for MHC proteins that are stable in the absence of peptide, such as HLA-DRB1Ã01:01 (DR1) [5], the focus of this study, peptide binding reactions proceed slowly because of most of the preparation adopts a peptide-averse conformation [6,7,8]. To alleviate these problems, photocleavable MHC-binding peptides were developed [9,10]. This strategy allowed generation of many different MHC-peptide complexes for interrogation of T cells [10,13] and for study of molecular aspects of the MHC-peptide interaction [14,15]
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