The activation of helper T cells by peptides bound to proteins of the class II Major Histocompatibility Complex (MHC II) is pivotal to the initiation of an immune response. The primary functional requirement imposed on MHC II proteins is the ability to efficiently bind thousands of different peptides. Structurally, this is reflected in a unique architecture of binding interactions. The peptide is bound in an extended conformation within a groove on the membrane distal surface of the protein that is lined with several pockets that can accommodate peptide side-chains. Conserved MHC II protein residues also form hydrogen bonds along the length of the peptide main-chain. Here we review recent advances in the study of peptide-MHC II protein reactions that have led to an enhanced understanding of binding energetics. These results demonstrate that peptide-MHC II protein complexes achieve high affinity binding from the array of hydrogen bonds that are energetically segregated from the pocket interactions, which can then add to an intrinsic hydrogen bond-mediated affinity. Thus, MHC II proteins are unlike antibodies, which utilize cooperativity among binding interactions to achieve high affinity and specificity. The significance of these observations is discussed within the context of possible mechanisms for the HLA-DM protein that regulates peptide presentation in vivo and the design of non-peptide molecules that can bind MHC II proteins and act as vaccines or immune modulators.

The homodimeric immunoreceptor NKG2D drives the activation of effector cells following engagement of diverse, conditionally expressed MHC class I-like protein ligands. NKG2D recognition is highly degenerate in that a single surface on receptor monomers binds pairs of distinct surfaces on each structurally divergent ligand, simultaneously accommodating multiple nonconservative ligand allelic or isoform substitutions. In contrast to TCR-pMHC and other NK receptor-ligand interactions, thermodynamic and kinetic analyses of four NKG2D-ligand pairs (MIC-A*001, MIC-B*005, ULBP1, and RAE-1beta) reported here show that the relative enthalpic and entropic terms, heat capacity, association rates, and activation energy barriers are comparable to typical, rigid protein-protein interactions. Rather than "induced-fit" binding, NKG2D degeneracy is achieved using distinct interaction mechanisms at each rigid interface.

Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are subsequently presented to T cells. Previous studies have shown that most of the residues required for binding of the chicken ovalbumin (Ova) 323-339 peptide to the I-A(d) MHC class II protein are contained within the shorter 325-336 peptide. This observation is somewhat inconsistent with the X-ray structure of the Ova peptide covalently attached to I-A(d) ( structure) in which residues 323 and 324 form binding interactions with the protein. A second register for the Ova(325-336) peptide is proposed where residues 326 and 327 occupy positions similar to residues 323 and 324 in the structure. Two Ova peptides that minimally encompass the and alternate registers, Ova(323-335) and Ova(325-336), respectively, were found to dissociate from I-A(d) with distinct kinetics. The dissociation rates for both peptides were enhanced when the His81 residue of the MHC beta-chain was replaced with an asparagine. In the structure the betaH81 residue forms a hydrogen bond to the backbone carbonyl of I323. If the Ova(325-336) peptide were also bound in the register, there would be no comparable hydrogen-bond acceptor for the betaH81 side chain that could explain this peptide's sensitivity to the betaH81 replacement. The Ova(323-335) peptide that binds in the register does not stimulate a T-cell hybridoma that is stimulated by Ova(325-336) bound in the alternate register. These results demonstrate that a single peptide can bind to an MHC peptide in alternate registers producing distinct T-cell responses.

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...