Dipeptides catalyze rapid peptide exchange on MHC class I molecules

Abstract

Peptide ligand selection by MHC class I molecules, which occurs by iterative optimization, is the centerpiece of immunodominance in antiviral and antitumor immune responses. For its understanding, the molecular mechanisms of peptide binding and dissociation by class I molecules must be elucidated. To this end, we have investigated dipeptides that bind to the F pocket of class I molecules. We find that they accelerate the dissociation of prebound peptides of both low and high affinity, suggesting a mechanism of action for the peptide-exchange chaperone tapasin. Peptide exchange on class I molecules also has practical uses in epitope discovery and T-cell monitoring.peptide exchange | MHC tetramers | tapasin | immunotherapy | dipeptides M HC class I proteins, which are expressed on the surface of nucleated cells of higher vertebrates, present intracellular peptides to cytotoxic T lymphocytes (CTLs). To bind to class I, a peptide must fulfill allotype-specific length and sequence requirements: Hydrogen bond networks that hold the N and C termini usually restrict peptide length to 8-10 amino acids, and side chains at defined positions (anchor residues) must fit into specificity pockets at the bottom of the binding groove (1).Cellular selection of antigenic peptides that conform to these requirements is an important step in immunodominance, the focusing of the adaptive immune system on a few epitopes in an immune response to viruses and cancer (2). This selection is thought to occur via iterative optimization, usually with the help of the peptide-exchange chaperone tapasin (3). Despite longstanding investigations, the molecular mechanism of peptide binding, dissociation, and tapasin-mediated exchange has remained elusive (4), in part because of the lack of structural information about peptide-free class I proteins.To explore the molecular mechanism of peptide binding and dissociation, we have searched for small molecules that can bind to class I and modulate the binding of high-affinity peptides. We have demonstrated previously that dipeptides of appropriate sequence bind to class I molecules, presumably to the F pocket, and support their folding (5), and we now show that they also dramatically accelerate the dissociation of prebound peptides and their exchange for exogenous peptides in a function analogous to that of tapasin. ResultsOn MHC class II molecules, short dipeptides that bind into the P1 pocket catalyze the exchange of prebound peptides for exogenous ones (6, 7). Because we found previously that certain dipeptides bind to and stabilize the MHC class I proteins H-2K b (K b ) and HLA-A*02:01 (A2) in the absence of high-affinity fulllength peptides (5), we asked whether such dipeptides also promote peptide exchange on MHC class I. We expressed recombinant A2 in Escherichia coli, folded it in vitro with the peptide NLVPMVATA, removed the free peptide by gel filtration, and added NLVPK FITC VATV (in which the lysine side chain is labeled with FITC in a position that does not interfere with binding to A2). The bi...

Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) Polymorphism Relevant to Inflammatory Disease Shapes the Peptidome of the Birdshot Chorioretinopathy-Associated HLA-A*29:02 Antigen*

“…Thus, ERAP2 might be particularly relevant in the context of low activity ERAP1 variants. A third possibility is that, since ERAP2 efficiently degrades 8-mers and shorter peptides, it might influence in this way the substrate-inhibition kinetics of ERAP1 trimming (20,38) or the affinity-dependent peptide exchange on the MHC-I molecule mediated by very small peptides (39,40).…”

TAPBPR mediates peptide dissociation from MHC class I using a leucine lever

“…To test whether the K22-D35 loop of TAPBPR was a functionally important region for mediating peptide selection, we first replaced all the residues in the loop with either glycine, alanine or serine, to produce a TAPBPR variant with a potentially functionless loop (TAPBPR Øloop )( Table 1). More subtle mutations of this loop were designed based on the work by Springer and colleagues, who demonstrated that dipeptides carrying long hydrophobic residues are able to bind to the peptide-binding groove of recombinant MHC I and enhance peptide dissociation [15]. Thus, we explored whether a leucine residue at position 30 of the mature TAPBPR protein, the only long hydrophobic residue within the entire loop, was involved in peptide exchange on MHC I.…”

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

“…The plate was incubated overnight at 37 °C with 5% CO 2 before the cells were transferred to 96-well U-bottomed plates and spun at 2000 rpm for 2 min. The cells were washed twice with PBS and lysed with 100 μl lysis buffer containing PBS with 0.5% Nonidet P40 (Sigma-Aldrich) and 0.15 mM Chlorophenol Red-ß-D-galactopyranoside (Sigma-Aldrich)26. The assay was incubated for a further 6 h at room temperature with absorbance readings at 570 nm recorded every 10 min in an Infinite Pro M200 plate reader (Tecan).…”