Journal of Molecular Biology
Crystal Structure Of MHC Class II I-Ab in Complex with a Human CLIP Peptide: Prediction of an I-Ab Peptide-binding Motif
Introduction
Class II major histocompatibility complex (MHC) molecules bind peptide fragments derived from exogenous protein sources and transport them to the cell surface for recognition by helper T cells.1 The invariant chain (Ii) plays several roles in class II MHC transport and antigen presentation.2 During the assembly of class II MHC molecules in the endoplasmic reticulum, a nine-chain structure is assembled from the association of three MHC αβ dimers with a single invariant chain trimer. The interaction of Ii with class II MHC molecules prevents the binding of extraneous peptides during transportation to the specialized endosomal compartments known as MIIC.3., 4., 5., 6. The cytoplasmic part of Ii contains an endosomal-targeting signal7., 8. that guides the transport of the complex through the Golgi apparatus and into the acidic endocytic MIIC compartments, where Ii is degraded in several stages.9., 10. The degradation intermediates, including lip22 (or LIP, ∼22 kDa), lip10 (or SLIP, ∼10 kDa), and CLIP, remain bound to the class II MHC molecules. Thus, complexes of class II MHC molecules with CLIP represent natural intermediates in the class II MHC processing pathway.11., 12., 13., 14., 15., 16., 17. CLIP is normally exchanged for an antigenic peptide in a reaction regulated by the class II MHC-like molecules HLA-DM/HLA-DO (in humans; H2-M/ H2-O in mice).18., 19., 20., 21. After binding peptides, class II MHC molecules are transported to the cell surface where their peptide contents are presented to CD4+ T-cells.
Three genetically distinct isotypes of class II MHC molecules are found in humans (HLA-DR, HLA-DQ, and HLA-DP), and two in mice (I-E and I-A). Previous work on class II MHC has focused on structural studies of the HLA-DR isotype22., 23., 24., 25., 26., 27., 28., 29., 30. and its murine homologue, I-E.31 However, recently the structures of a number of the I-A alleles have been solved, including those of I-Ad,32 I-Ak,33 and I-Ag7.34., 35. Class II MHC share a common structural topology. In particular, the peptide-binding groove is formed by two long antiparallel α-helical segments that sit on top of and traverse an eight-stranded antiparallel β-sheet. Within the groove, pockets and clefts accommodate the peptide side-chains of a certain size and charge in an allele-specific way. In general, class II peptides use a nine residue “core” P1–P936 to interact with the peptide-binding groove. Due to the distinct nature and arrangement of residues in the peptide-binding groove, each class II MHC molecule has its own unique preference for peptides.
CLIP represents a set of nested peptides derived from the residues 81–104 of Ii.11., 13. The core-binding region of CLIP for most class II MHC molecules is a nine residue fragment (MRMATPLLM, 91–99 of Ii).37., 38. The CLIP peptides from human (hCLIP) and mouse (mCLIP) share the same core sequence, but differ in their flanking regions (Figure 1A). Of particular note, except for residues 92 and 95, is that all other residues in the core region are non-polar, including three Met residues at positions 91, 93, and 99. CLIP can bind to MHC class II with an affinity that can vary by up to several orders of magnitude.39., 40., 41. However, CLIP binds in the peptide-binding groove in a way similar to that of other antigenic peptides.24., 39., 40., 41., 42., 43., 44. The Met residues in the CLIP sequence, in particular, Met91, have been shown to be important for binding in many allelic variants of MHC class II.39., 40., 41.
I-Ab/CLIP complexes are observed on the cell surface of both wild-type and presentation-deficient antigen-presenting cells.11., 38. To facilitate studies on the function of I-Ab in the immune system, various monoclonal antibodies have been generated with specificity for I-Ab.38., 45., 46., 47., 48. The recognition of I-Ab/CLIP complexes on the surface of normal and mutant cells has been probed by T-cells and by monoclonal antibody (mAb) 30-2, and found it to be indistinguishable from that of normal antigenic peptides.49 T-cell hybridomas specific for human I-Ab/CLIP complexes recognize, in particular, the Gln residue at position 100 (P10) of human CLIP, while mAb 30-2 recognizes the Lys residue at position 90.49
Furthermore, in H2-M deficient mice, CLIP is observed to be bound by the vast majority of surface-expressed I-Ab molecules. In addition, significantly reduced numbers of mature CD4 T-cells are observed in these mice compared to wild-type.50., 51. Human Ii transgenic mice crossed with Ii/H2-M double deficient mice display a similar skewing in their peptide repertoire (when bound to I-Ab), as well as a defect in the development of CD4 T-cells (A.R., unpublished results). These mice have become a critical model for evaluating the role of peptide specificity in positive selection. The elucidation of the I-Ab/CLIP structure in this study should promote a greater understanding of the role of both peptide and MHC in the positive selection of the TCR.
Section snippets
Structure determination
I-Ab/CLIP was expressed in Drosophila S2 cells and purified by Ni-NTA affinity chromatography and several steps of traditional chromatography (see Materials and Methods). Crystals were obtained after enzymatic cleavage of the leucine zipper-polyhistidine tail, and belong to the space group C2 with unit cell dimensions a=63.65 Å, b=88.07 Å, c=83.74 Å, and β=92.48°. The atomic coordinates of I-Ak (PDB accession code 1IAK) were used as the starting model for molecular replacement. The structure has
Discussion
The crystal structure of I-Ab has provided insights into the peptide-binding properties of this I-A allele, which hitherto had remained controversial despite extensive peptide-binding studies.56., 67. Although spatially the P1 and P9 pockets are capable of accommodating any sized residue, I-Ab displays a distinct preference at these positions for hydrophobic residues. Pockets 4 and 6 are smaller than those in other I-A molecules and may play an important role in the allele-specificity of
Cloning of the I-Ab constructs
The expression constructs for I-Ab/CLIP were generated in a similar way to the constructs described for I-Ad/OVA.72., 73. The α subunit construct was designed to express a fusion polypeptide containing the extracellular domain of the mature I-Ab α subunit (residues 1–178) attached to a carboxyl tail consisting of a thrombin cleavage site, an acidic leucine zipper peptide, and a hexahistidine tail. The β subunit construct was designed to express a fusion polypeptide containing CLIP, covalently
Acknowledgements
We thank Drs Xiaoping Dai, Samantha Greasley, and Andreas Heine for support during synchrotron data collection and data processing, Marc Elsliger for assistance in computational analysis, and Randy Stefanko for excellent technical help in large-scale cell culture and protein production. This work was supported by National Institutes of Health grant CA-58896 (to I.A.W.) and DK55037 (to L.T.).
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