Comparison of crystallized MHC class II⅐peptide complexes has revealed that, in addition to pocket interactions involving the peptide side chains, peptide binding to MHC class II molecules is characterized by a series of hydrogen bonds between genetically conserved amino acid residues in the class II molecule and the main chain of the peptide. Many class II⅐peptide structures have two sets of symmetrical hydrogen bonds at the opposite ends of the class II antigen-binding groove (-His-81, -Asn-82 vs. ␣-His-68, ␣-Asn-69). In this study, we alter these peripheral hydrogen bonds and measure the apparent contribution of each to the kinetic stability of peptide⅐class II complexes. Single conservative amino substitutions were made in the I-A d protein to eliminate participation as a hydrogen bonding residue, and the kinetic stability of a diverse set of peptides bound to the substituted I-A d proteins was measured. Although each hydrogen bond does contribute to peptide binding, our results point to the striking conclusion that those hydrogen bonds localized to the amino terminus of the peptide contribute profoundly and disproportionately to the stability of peptide interactions with I-A d . We suggest that the peripheral hydrogen bonds at the amino terminus of the bound peptide that are conserved in all class II⅐peptide crystal structures solved thus far form a cooperative network that critically regulates peptide dissociation from the class II molecule.M ajor histocompatibility complex (MHC) class II molecules are composed of two noncovalently associated, polymorphic transmembrane proteins, termed ␣ and , of approximate molecular mass of 33 kDa and 28 kDa, respectively. They interact with T cell receptors to provoke an antigen-specific CD4 ϩ T cell response. Crystal structures of class II molecules have provided insight into the structural elements that control interactions with peptide. Both polymorphic and genetically conserved amino acid residues make contacts with the bound peptide (1-7). Most of the polymorphic residues face the interior of the peptide-binding groove, and thus contribute to allele-dependent peptide binding. Similar to what has been observed in class I molecules, MHC class II molecules appear to have ''pockets'' that are capable of binding to peptide side chains. In addition to these pocket interactions, MHC class II molecules bind peptide through a series of hydrogen bonds between genetically conserved amino acid residues in the class II molecule and the main chain of the peptide. These conserved residues bind at relatively regular intervals throughout the length of the peptide, and apparently stabilize the peptide in a fairly fixed polyproline type II conformation throughout the length of the peptide-binding pocket (3).One striking aspect of class II⅐peptide structures noted in murine complexes (6, 7) are two sets of symmetrical hydrogen bonds at the opposite ends of the binding pocket (Fig. 1). The hydrogen bonds contributed by -His-81 and -Asn-82 are localized to the bound peptide's amino...