Empty Class II Major Histocompatibility Complex Created by Peptide Photolysis Establishes the Role of DM in Peptide Association

Abstract

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.Major histocompatibility complex (MHC) 5 molecules are cell surface proteins that present peptides to antigen-specific receptors on T cells. The Class II MHC products are specialized in sampling endosomal compartments to acquire these peptides. Delivery of newly synthesized and assembled Class II MHC proteins to endo-lysosomal compartments is assured by means of the transient association of the MHC ␣␤ heterodimer with the invariant chain, a protein endowed with both chaperone function and an address code (1, 2). In endosomal compartments, the invariant chain is destroyed, yielding a Class II MHC product occupied with an invariant chain-derived remnant, the CLIP peptide (3-5). The HLA-DM (DM) molecule, itself incapable of binding peptide, facilitates replacement of CLIP with antigenic peptides (6 -10). The action of DM is not limited to the Class II MHC/CLIP complex and extends to Class II MHCpeptide complexes more generally. Such editing by DM favors presentation to CD4 T cells of those peptides that are most resistant to peptide displacement by DM (9,(11)(12)(13)(14)(15)(16)(17).DM is a membrane-anchored heterodimer that belongs to the extended family of proteins with a MHC fold but lacks a functional peptide-binding groove (18,19). Mutagenesis experiments identified lateral surfaces on DM and DR molecules that are involved in the interaction between the two proteins. On the DR side, these mutations span the entire length of the ectodomain and are localized to the ␣1 and ␤2 domains (20). On the DM side, an extended interaction surface has been mapped that also spans the entire length of the ectodomain (21). These data support a model in which lateral interactions between DM and DR molecules induce a conformational change that destabilizes the DR-bound peptide. It has been proposed that DM disrupts one or severa...

Promiscuous binding of extracellular peptides to cell surface class I MHC protein

“…The k den found here correspond to t 1/2 values of 38-88 min (at 37 8C). These values (∼50 min on average) (Table S1) are similar to the only other t 1/2 (55 min) for an empty MHC of which we are aware; the latter was found for a human class II MHC, HLA-DR [calculated by Kevin Fowler from data in the work by Grotenbreg et al (32) using a model that differed considerably from the model used here]. Whether these unexpectedly long-lived empty MHCs found for one mouse class I and one human class II MHC are representative of MHC proteins in general remains to be seen.…”

“…The procedures used to measure k on for peptide binding to ostensibly empty MHC molecules have varied widely, and the reported association rate constants have spanned a wide range, roughly from 10 2 to 10 6 M −1 s −1 . Many reported values are at the low end of this range (21) (SI Text, R16 and R17), similar to those values found here and also for peptide binding to a class II MHC (32). When expressed in conventional units (M −1 s −1 ) rather than in the units (M −1 h −1 ) used in Table 1, we found 4,900 M −1 s −1 for OVA-K b and 217 M −1 s −1 for SIY-K b (Table 1).…”

MHC Class II Antigen Presentation by Dendritic Cells Regulated through Endosomal Sorting

“…During such peptide exchange, the MHCII-binding groove is temporarily empty, a state that is rather unstable and prone to aggregation (Germain and Rinker 1993;Rabinowitz et al 1998). DM prevents this aggregation and stabilizes empty MHCII molecules, keeping them in a peptide-receptive state (Kropshofer et al 1997;Grotenbreg et al 2007;Anders et al 2011). This important DM-catalyzed editing process favors the generation of high-affinity pMHCII complexes and is facilitated by the acidic milieu in late endosomes.…”

Stability Screening of Arrays of Major Histocompatibility Complexes on Combinatorially Encoded Flow Cytometry Beads

“…1). After UV irradiation, the peptide fragments are believed to be liberated from the complex (5)(6)(7)(8)12). The uncaged complex can be rescued from degradation when UV-mediated cleavage is performed in the presence of a ligand capable of stabilizing the MHC molecule (Fig.…”

“…Cell-free biophysical methods that employ soluble complexes purified to homogeneity have the advantage of directly measuring the molecular interaction between the peptide and MHC without the confounding effects of simultaneous expression of multiple MHC variants in a cellular context. Limitations on the expeditious and high throughput generation of collections of recombinantly produced pMHC products have largely been resolved with the introduction of conditional ligands for class I human leukocyte antigens (HLAs), (5-7) murine MHC, (5, 8 -11), and class II MHC molecules (12) and have been exploited for an ELISA-based MHC stability assay (6,13). Nevertheless, methods that employ cell lines defective in antigen presentation, where the exogenous addition of peptide can measurably restore MHC surface expression, such as the murine RMA-S (14,15) or human T2 lines transfected with an MHC of choice (16), remain unceasingly popular.…”