The Crystal Structure of H-2Db Complexed with a Partial Peptide Epitope Suggests a Major Histocompatibility Complex Class I Assembly Intermediate

Abstract

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324 -332, 1 FAPGNYPAL 9 ) to promote its folding in vitro of H-2D b . We found that H-2D b can be stabilized by the pentapeptide 5 NYPAL 9 , which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D b molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove. Major histocompatibility complex (MHC)5 class I molecules have evolved to present peptide epitopes of 8 -10 amino acids to cytotoxic T cells. Many MHC class I⅐peptide structures have now been solved by x-ray crystallography, and they all have a common tertiary structure (1). The structure consists of a polymorphic heavy chain (HC) and a nonpolymorphic light chain ␤2-microglobulin (␤2-m), noncovalently associated to form a molecule with two membrane proximal Ig-like domains (the ␣3 and ␤2-m domains) that support a membrane distal ␣1-␣2 "superdomain." The peptide-binding site is formed by a deep cleft between two ␣-helices in this superdomain. Antigenic peptides are always bound in the same orientation, with their N and C termini lying buried deep in pockets that define the ends of the peptide-binding groove (the A and F pockets, respectively). In addition, so-called "anchor residues" make allele specific interactions with polymorphic class I residues located deep inside the binding groove, in "specificitydetermining pockets."The peptide⅐MHC class I complex is formed in the endoplasmic reticulum (ER) and marks the end point of antigen processing (2). During antigen processing, proteins are unfolded and partially hydrolyzed in the cytoplasm, and the resulting polypeptides (of between 8 and 40 amino acids) are translocated across the ER membrane by the transporter associated with antigen processing. Once in the ER, some long peptide epitope precursors can undergo further trimming by the aminopeptidase ERAAP (3, 4) and are selected for assembly with newly synthesized MHC class I molecules that is dependent on their interaction with cofactor molecules such as calreticulin, tapasin, and ERp57. The process results in the preferential release from the ER of class I molecules presenting peptides that bind stably. Recent evidence suggests that this selection of high affinity peptides in vivo may occur by a mechanism that is more complex and controlled than simple competition between potential ligands for binding to class I in the ER (2) and may involve editing of the MHC-bound peptide repertoire in the early secretory pathway of antigen-presenting cells.It is not known whether the loading or editing of class I MHC peptide cargo...

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

“…This is consistent with published literature, because the human ␤2m:H-2D b chimeric complex contains more hydrogen bonds between the heavy and light chain and is more stable than its exclusively murine counterpart (22). An x-ray crystal structure has also shown that the H-2D b complex was unexpectedly stabilized by a small pentapeptide fragment, indicating that full occupancy of the peptidebinding groove is a less stringent requirement for stability of this complex (31). Fortunately, variables such as incubation time, temperature, and buffer conditions can provide additional control over the assay, although experience with other HLA variants suggests that by and large the standard peptide exchange conditions sufficiently destabilize MHC molecules (data not show) making the stability of the H-2D b complex a notable exception.…”

Dipeptides promote folding and peptide binding of MHC class I molecules

“…Importantly, experiments in vivo and in vitro have shown that, in the presence of the PLC, peptide loading is iterative, i.e., class I molecules first bind suboptimal peptides (peptides that are too long, too short, or do not have the requisite anchor residues) and gradually exchange them for higher-affinity ones (10,11). The idea that class I molecules can initially fold with such suboptimal peptides is indeed supported by the crystal structures of several class I molecules with octamer or pentamer peptides (12,13).…”

“…Shorter peptides that correspond to fragments of high-affinity peptides have previously been crystallized in complex with class I molecules (12,13). We therefore hypothesized that short peptides may support the folding of class I molecules but may then be easily removed because of their low affinity for class I.…”

A role for UDP-glucose glycoprotein glucosyltransferase in expression and quality control of MHC class I molecules

“…Molecular dynamic modeling suggests that the region that binds the peptide C terminus is more flexible in the empty state than the corresponding region interacting with the peptide N terminus (31). The empty N-terminal region adopts a "preformed" conformation that is similar to the occupied state (31) and can be largely satisfied by solvents like water in crystal structures (24,26). In contrast, the empty C-terminal region shows much more fluctuation (31), and it has been proposed that the interaction between the F pocket in the C-terminal region, which is near N86, and the C terminus of the bound peptide is most critical for stabilizing MHC class I molecules (26).…”