Speculation regarding the mechanism by which tapasin regulates peptide binding to MHC class I molecules has often centered on comparisons with that of HLA-DM, which performs a similar function for MHC class II (Busch et al., 2005
; Sadegh-Nasseri et al., 2008
). While superficially the processes are similar, tapasin function is clearly more complex in that it operates in cooperation with a number of other components, including its heterodimeric partner, ERp57. The structure of the tapasin/ERp57 heterodimer revealed here increases our appreciation for the differences between the two; DM is a structural homologue of class II molecules themselves (Fremont et al., 1998
; Mosyak et al., 1998
), while tapasin lacks any resemblance to DM or MHC molecules except that all are members of the immunoglobulin superfamily. Determination of the tapasin/ERp57 structure allows us to address several outstanding questions regarding the architecture and function of the PLC. In particular, we evaluate two central issues. First, what is the mechanism and function of covalent sequestration of ERp57 by tapasin? Second, how do tapasin and the other PLC components interact with empty MHC class I molecules to facilitate their stabilization and loading with high affinity peptides?
We previously postulated that the disulfide bond formed between tapasin and ERp57 is preserved as a consequence of non-covalent interactions (Peaper et al., 2005
). The structure provides no evidence that the escape pathway is inhibited by perturbation of the a
domain active site. Instead it appears that the heterodimer is stabilized by the association of tapasin with both of the active sites of ERp57. This finding has several implications. It suggests that tapasin has evolved and acquired these non-covalent interactions as the primary means for stabilizing what would otherwise be a transient mixed disulfide between ERp57 and substrate. It is also consistent with the evidence that the stable, covalent association with ERp57 has a direct effect on the activity of tapasin (Wearsch and Cresswell, 2007
) and speculation that it might affect tapasin conformation. Lastly, the association of tapasin with both active sites of ERp57 has strong implications regarding the role of ERp57 in the PLC.
ERp57 functions in the early oxidative folding of the MHC class I heavy chain (Zhang et al., 2006
), and some studies have also suggested a role for its enzymatic activity within the PLC, i.e. in reduction/oxidation of the disulfide bond at the base of the MHC class I peptide binding groove (Dick et al., 2002
; Lindquist et al., 2001
; Santos et al., 2007
). In the crystal structure, however, both ERp57 active sites are engaged in contacts with tapasin and inaccessible to the MHC class I molecule (). While we cannot exclude that ERp57 may assume an alternative conformation within the PLC, the structures described here suggest that tapasin-associated ERp57 does not exhibit redox activity. In further support of this conclusion, we recently found by mutational analysis that the cysteines in the ERp57 a
′ catalytic motif are not required for efficient MHC class I peptide loading (Peaper and Cresswell, 2008
). Although the structure is not inconsistent with the conclusion by Dick and co-workers (Kienast et al., 2007
) that tapasin inhibits the redox activity of ERp57 to prevent reduction of class I heavy chains, we favor an alternate explanation for the role of tapasin/ERp57 conjugation and a positive role for ERp57 in peptide loading. Other studies (Garbi et al., 2006
; Peaper and Cresswell, 2008
; Wearsch and Cresswell, 2007
) better support a model in which tapasin has adapted the quality control machinery by covalently sequestering ERp57 in order to enhance the recruitment and stability within the PLC of empty MHC class I molecules associated with calreticulin, which interacts independently with the class I glycan and the bb
′ domains of ERp57 (Helenius and Aebi, 2004
Two long helices, α1 and α2, form the walls of the MHC class I peptide binding groove. Molecular dynamics simulations as well as the crystal structure of a peptide-free MHC class I-like molecule (Olson et al., 2005
; Zacharias and Springer, 2004
) support the proposal that in the absence of peptide, the binding groove adopts an “open” conformation characterized by mobility in the N-terminal segment of helix α2, α2-1 (Elliot, 1997
). Tapasin is thought to stabilize a peptide-receptive, open conformation of MHC class I, and mutations on the same side as α2-1 affect tapasin binding ()(Carreno et al., 1995
; Peace-Brewer et al., 1996
; Lewis & Elliot, 1998
; Yu et al., 1999
). We suggest that the conserved, functionally important surface of tapasin that we have identified serves to stabilize helix α2-1 while the binding groove maintains an open conformation. Both tapasin and MHC class I are tethered to the ER membrane by C-terminal transmembrane segments, and, given this restraint, the two molecules can be juxtaposed so that the conserved surface in tapasin is accessible to α2-1 (). The shallow trough within which the conserved residues are located () could easily accommodate and stabilize a short helix such as α2-1. Within the PLC, the MHC class I molecule would then remain primarily in an open conformation until peptide binds with high affinity, locking it into the “closed” conformation observed in peptide-bound structures. This weakens the interaction with tapasin, releasing the class I molecule from the PLC and allowing it to move to the plasma membrane for recognition by CD8+ T cells.
In silico model of the lumenal subcomplex of the PLC
Atomic resolution structures of the peptide-bound form of MHC class I (we used PDB ID 1DUY (Khan et al., 2000
) for HLA-A*0201) and of the calreticulin homolog calnexin are available (PDB ID 2JHN (Schrag et al., 2001
)), as is a vast body of biochemistry regarding the interactions of the MHC class I molecule with tapasin and calreticulin and of calreticulin/calnexin with ERp57 (Wright et al., 2004
). Combining these data with the tapasin/ERp57 structure and the MHC class I interaction site identified here allowed us to generate a rational model of the sub-complex of the PLC containing the lumenal components (). Class I heavy chain residues known to interact with tapasin (Carreno et al., 1995
; Peace-Brewer et al., 1996
; Lewis & Elliot, 1998
; Yu et al., 1999
) are modeled at the tapasin/class I interface, and helix α2-1 is near the conserved, functionally important surface of tapasin we have identified (). For calreticulin, we have substituted the calnexin structure but with an appropriately shortened proline-rich P-domain. The lectin-binding site of calreticulin (Thomson and Williams, 2005
) is placed proximal to the N-linked glycan at Asn86 of the MHC class I heavy chain which, in the monoglucosylated form, is known to interact with calreticulin (Harris et al., 1998
; Wearsch et al., 2004
). The P-domain is flexible (Ellgard et al., 2002
), and it was incorporated into the model so that its tip is adjacent to the basic surface of ERp57 b
′ domain known to interact with this region of calnexin and calreticulin (Kozlov et al., 2006
; Russell et al., 2004
). The conformation of the P-domain indicated in the model is one of a number that are possible, but in all likelihood it forms an arm that encloses the empty MHC class I peptide binding groove. This could protect the incompletely assembled MHC class I molecule from reduction by free, non-PLC-associated, ERp57, or other thiol oxidoreductases in the ER such as PDI. It may also protect the still improperly folded empty peptide binding groove from recognition by other ER chaperones that might target the peptide-free MHC class I molecule for degradation.
Additional evidence in support of the model structure indicated in is provided by an MHC class I mAb epitope that is the only one known to be expressed and readily accessible on MHC class I molecules present in the PLC. The 64-3-7 epitope has been used to define the ‘open’ conformation of class I putatively present in the PLC. It is present in H2-Ld
molecules, can be transferred to other MHC alleles, and corresponds to residues 46–52 in the MHC class I heavy chain (Hansen et al., 2005
). This sequence, which lies towards the N-terminal end of the α1 helix, would be easily available to an antibody in the structure proposed (). Tapasin residue 191, which is part of the PaSta2 epitope obscured by MHC class I binding, is buried in the MHC class I/tapasin interface.
Data from Shastri and co-workers (Kanaseki et al., 2006
), have argued that binding of N-terminally extended peptides to MHC class I molecules followed by trimming by the enzyme ERAAP/ERAP-1 is likely to occur during peptide loading. In the MHC class I orientation shown in the model in , N-terminally extended peptides transiently associated with MHC class I molecules within the PLC would be accessible to ERAAP/ERAP-1. This suggests that such peptide trimming need not be restricted to MHC class I molecules that are freely diffusing in the ER membrane.
More than a decade has passed since the discovery of tapasin and the PLC, and yet much work remains before we fully understand the mechanisms by which they facilitate peptide loading and editing. A major obstacle to designing experiments to probe this mechanism has been the lack of structural information regarding the PLC and, in particular, the MHC class I-specific chaperone tapasin. Thus, the structure of the tapasin/ERp57 heterodimer and the model for the lumenal sub-complex of the PLC that we present here is not only a major step toward better understanding the architecture/function of the PLC, but, equally importantly, will serve as a powerful springboard for further mechanistic probing.