Stability of empty and peptide-loaded class II major histocompatibility complex molecules at neutral and endosomal pH: Comparison to class I proteins

Abstract

The structure and thermal stability of empty and peptide-filled forms of the murine class II major histocompatibility complex (MHC) molecule I-E k were studied at neutral and mildly acidic pH. The two forms have distinct circular dichroic spectra, suggesting that a conformational change may accompany peptide binding. Thermal stability profiles indicate that binding of peptide significantly increases the thermal stability of the empty heterodimers at both neutral and mildly acidic pH. Free energies calculated from these data provide a direct measure of this stabilization and show that the empty form of I-E k is significantly more stable than that of class I MHC proteins. Furthermore, for the two MHC class II proteins that were analyzed (I-E k and I-A d ), thermal stability was not significantly altered by acidification. In contrast, of four class I MHC molecules studied, three have shown a significant loss in complex stability at low pH. The marked stability exhibited by their empty form, as well as their resistance to low pH, as observed in this study, correlate well with the ability of class II MHC molecules to traverse and bind peptides in acidic endosomal vesicles.Class I and class II major histocompatibility complex (MHC) molecules are heterodimeric cell surface glycoproteins that bind antigenic peptides and display them for surveillance by T lymphocytes (1). The two MHC classes have a similar structure with two membrane-proximal immunoglobulin-like constant domains and a membrane-distal peptide-binding groove formed by two ␣-helices atop an eight-stranded ␤-sheet floor (2, 3). The interaction between the MHC molecule and the peptide not only forms the basis for the heterodimer function in antigen presentation but also plays a decisive role in its thermodynamic stability. For class I molecules, the presence of an appropriate peptide has been shown, in most circumstances, to be imperative for successful folding and surface expression at physiological temperature (1, 4-8). The free energy contributed by peptide binding to empty class I K d heterodimers has been determined (9) and the binding energies contributed by peptide contacts at anchor positions (10-13) and at the peptide NH 2 and COOH termini (8) have been reported for several alleles. The stability of class II molecules also depends on the peptide (1, 14), though apparently to a lesser extent than class I molecules (15). In addition, the binding of peptides to class II proteins is pH-dependent. For most class II alleles, peptide binding is enhanced at low pH (1,(16)(17)(18)(19)(20), consistent with the pH of the endosomal-like compartment (pH Ϸ5.0) where peptide loading takes place. At that pH, it has been suggested that the molecules adopt an alternate ''open'' conformation that facilitates peptide binding (21-24) and that has been proposed to be less stable than the form adopted at neutral pH (21,(23)(24)(25).In spite of these observations, direct quantitative data regarding the energetic consequences of peptide binding or alteration in the p...

Peptide-Independent Folding and CD8αα Binding by the Nonclassical Class I Molecule, Thymic Leukemia Antigen

“…The T m value of 54°C is similar to the values (54 and 57°C) reported for H-2K d bound to specific peptide ligands (35,38). Most class Ia molecules, like HLA-A2 in the present study, fail to assemble in the absence of appropriate peptides, indicating that peptide is an integral component of the properly folded molecules.…”

“…A second transition with T m Ͼ60°C characterized by a sign reversal of the CD signal probably represents unfolding of free ␤ 2 m (35,40). The 54°C T m of empty T18 d is similar to that reported for H-2K d peptide complexes (54 -57°C) (35,38) and greater than that reported for the class Ib protein T10 (49°C) (40), which does not bind peptides. Thus empty T18 d molecules are relatively stable and folding the protein in the presence of peptide does not increase its stability.…”

“…Far-UV CD spectroscopy has been used to analyze the structure and thermal stability of a variety of classical and nonclassical MHC molecules (17,(35)(36)(37)(38)(39)(40). The far-UV spectra of T18 d are shown in Fig.…”

A Conformational Change in the Human Major Histocompatibility Complex Protein HLA-DR1 Induced by Peptide Binding

“…Mildly acidic pH (4.5-6.5) decreases the SDS stability of the protein and increases its ability to bind the fluorescent probe ANS (8-amino-1-naphthalenesulfonic acid) (23,24), providing evidence for a pH-induced structural change in class II MHC proteins. Recently, direct physical comparison of empty and peptide-loaded MHC has been reported for I-E k (25), where small changes in circular dichroism spectra observed at mildly acidic pH were interpreted as a manifestation of a peptide-dependent conformational change. In that study, the peptide-bound form exhibited a steeper thermal denaturation profile than the empty protein, which is suggestive of a looser or extended conformation in the empty protein, and indicating a possible similarity to the "floppy" species described above.…”

The A′ and F′ Pockets of Human CD1b Are Both Required for Optimal Presentation of Lipid Antigens to T Cells

“…It has become evident that MHC I binds peptides through the formation of hydrogen bonds between the pockets of MHC I and the amino acid sidechains of bound peptides (18,19,(22)(23)(24)(25)(26)(27)(28). Additional stabilizing interactions occur between the charged peptide N and C termini and the ends of the MHC I groove.…”