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...