Insights into MHC class I peptide loading from the structure of the tapasin-ERp57 thiol oxidoreductase heterodimer

Abstract

Tapasin is a glycoprotein critical for loading major histocompatibility complex (MHC) class I molecules with high-affinity peptides. It functions within the multimeric peptide-loading complex (PLC) as a disulfide-linked, stable heterodimer with the thiol oxidoreductase ERp57, and this covalent interaction is required to support optimal PLC activity. Here, we present the 2.6 A resolution structure of the tapasin-ERp57 core of the PLC. The structure revealed that tapasin interacts with both ERp57 catalytic domains, accounting for the stability of the heterodimer, and provided an example of a protein disulfide isomerase family member interacting with substrate. Mutational analysis identified a conserved surface on tapasin that interacted with MHC class I molecules and was critical for peptide loading and editing functions of the tapasin-ERp57 heterodimer. By combining the tapasin-ERp57 structure with those of other defined PLC components, we present a molecular model that illuminates the processes involved in MHC class I peptide loading.

Figure 1. The structure of the tapasin/ERp57 heterodimer

(A) Ribbon diagram with ERp57 in white. The N-terminal domain of tapasin is a fusion of a β-barrel (green) and an Ig-like domain (yellow); the C terminus is an Ig-like domain (blue). (B) Sequence alignment for tapasin. Residues in humans, mice, chickens, and zebrafish that are identical/similar are red/orange. Residues within 4.5 Å of an ERp57 atom are boxed in black. Secondary structure elements are labeled. Mutations that interfere with MHC class I interaction and function are indicated (TN3-TN6).

Figure 2. Interactions between tapasin and ERp57

(A) Tapasin residues within 4.5 Å of an ERp57 atom are labeled. (B) ERp57 has been pulled away from tapasin and rotated 180° about a vertical axis to expose the tapasin interaction surface. Residues at the tapasin interface are labeled. Contacts between tapasin and ERp57 are listed in Table S1. (C) Sequence alignment of ERp57 domains a and a′ and equivalent domains of other ER-resident PDIs. Residues in ERp57 within 4.5 Å of a tapasin atom are boxed green (domain a) or yellow (domain a′). Catalytic motifs are red. Secondary structure is indicated. (D) Domains a from yeast PDI (PDBID 2B5E) and ERp57 were superimposed. The catalytic motif in domain a of ERp57 (white) is not distorted when tapasin (green) is bound as compared with the equivalent motif in yeast PDI (red). Disulfide bonds are yellow.

Figure 3. A conserved surface in tapasin is important for PLC assembly/function

(A) Space filling representation of tapasin/ERp57 with surface residues colored red/orange as in Figure 1B. For underlined residues, the side chain forms part of the protein core. The complex is oriented as in Figure 1A. (B) The complex has been rotated 180° around a vertical axis. (C) Residues that were mutated are colored (Table 2). Mutations that affect PLC assembly/function are colored in shades of red. Others are blue/green.

Figure 4. Characterization of tapasin mutants

(A) In vitro assay for the association of MHC class I molecules with wild type (WT) and mutant tapasin/ERp57 conjugates. Digitonin extracts from .220. B*0801 cells were incubated with 250 nM of the indicated recombinant conjugates immobilized on beads. Samples were washed, analyzed bv SDS-PAGE and subjected to immunoblotting (IB) for the MHC class I heavy chain. Data are representative of at least 3 independent experiments. (B) In vitro assay comparing the ability of the recombinant tapasin/ERp57 conjugates to facilitate MHC class I peptide loading. Extracts were prepared from .220. B*0801 cells and incubated with 250 nM recombinant conjugate and [¹²⁵I]-NP(380-387L). Immunoprecipitation of MHC class I molecules were then performed with the W6/32 Ab and peptide loading was measured by gamma counting. Activities were normalized to that of the wild type tapasin/ERp57 conjugate. Data are an average of 2 independent experiments +/− the S. E. M. (C) The ability of the indicated tapasin constructs to restore HLA-B*4402 expression in tapasin-deficient cells was analyzed by FACS. .220. B*4402 cells transduced with the indicated constructs were stained with anti-HLA-A, B, C and cell surface expression was quantitated for EGFP+ cells. Data are representative of 2 independent experiments. (D) PLC composition in transduced .220. B*4402 cells. Immunoprecipitations were performed from digitonin extract, analyzed by non-reducing SDS-PAGE, and transferred to membranes for immunoblotting with Abs to TAP and MHC class I heavy chain. (E) The PaSta2 mAb does not bind to MHC class I-associated tapasin. Extracts of .220. B*0801 cells were supplemented with the indicated concentrations of purified tapasin/ERp57 conjugate and subjected to immunoprecipitation with either PaSta1 or PaSta2 and immunoblotting for the MHC class I heavy chain. (F) The TN7 mutation of tapasin eliminates recognition by PaSta2. Extracts were prepared from either uninfected Sf21 cells (−) or those infected with baculovirus for the indicated tapasin/ERp57 conjugates were immunoprecipitated with either PaSta1 or PaSta2 coupled beads and analyzed by non-reducing SDS-PAGE. Proteins were visualized by staining with Coomassie Blue.

Figure 5. In silico model of the lumenal subcomplex of the PLC

Structures of tapasin (grey) and MHC class I (green) are juxtaposed in (A, B), so that residues important for their interaction are at or near the protein interface. (A) Mutations in tapasin (space filling model) that affect PLC assembly are colored as in Figure 3 and Table 2. The heavy chain is dark green and β₂m is lighter. Helices α1, α2-1, α2-2 that define the MHC class I peptide binding groove are indicated, and a red line marks the groove. (B) The MHC class I molecule (space filling model) is juxtaposed with tapasin. Residues in the heavy chain that are important for interactions with tapasin (Carreno et al., 1995; Peace-Brewer et al., 1996; Lewis & Elliot, 1998; Yu et al., 1999) are highlighted yellow. (C) Side view of the assembled sub-complex, with tapasin, ERp57, MHC class I, and calreticulin. Residues in the ERp57 b′ domain that interact with the calreticulin P-loop are blue (Kozlov et al., 2006; Russell et al., 2004). The 64-3-7 epitope of MHC class I heavy chains (H2-L^d) that is accessible in the PLC is white. (D) Top view of the assembled sub-complex. The MHC class I molecule is shown with a semi-transparent surface. The site of glycosylation at Asn86 in the heavy chain is indicated. This glycan is bound in the calreticulin glucose-binding pocket. Calreticulin residues important for glucose binding are labeled blue (Thomson and Williams, 2005).