Cooperativity during the formation of peptide/MHC class II complexes

To generate an effective immune response, class II major histocompatibility complex molecules (MHCII) must present a diverse array of peptide ligands for recognition by T lymphocytes. Peptide/MHCII complexes are stabilized by hydrophobic anchoring of peptide side chains to pockets in the MHCII protein and the formation of hydrogen bonds to the peptide backbone. Many current models of peptide/MHCII association assume an additive and independent contribution of the interactions between major MHCII pockets and corresponding side chains in the peptide. However, significant conformational rearrangements occur in both the peptide and MHCII during binding. Therefore, we hypothesize that peptide binding to MHCII could be viewed as a folding process in which both molecules cooperate to produce the final conformation. To directly test this hypothesis, we adapt a serial mutagenesis strategy to study cooperativity in the interaction of the human MHCII HLA-DR1 and a peptide derived from influenza hemagglutinin. Substitutions in either the peptide or HLA-DR1 that are predicted to interfere with hydrogen bond formation show cooperative effects on complex stability and affinity. Substitution of a peptide side chain that provides a hydrophobic contact also contributes to the cooperative effect, suggesting a role for all energetic sources in the folding process. We propose that cooperativity throughout the peptide-binding groove reflects the folding of segments of the MHCII molecule into helices around the peptide with a concomitant folding of the peptide into a polyproline helix. The implications of cooperativity for peptide/MHCII structure and epitope selection are discussed.     

Specific non-native hydrophobic interactions in a hidden folding intermediate: implications for protein folding

Structures of intermediates and transition states in protein folding are usually characterized by amide hydrogen exchange and protein engineering methods and interpreted on the basis of the assumption that they have native-like conformations. We were able to stabilize and determine the high-resolution structure of a partially unfolded intermediate that exists after the rate-limiting step of a four-helix bundle protein, Rd-apocyt b(562), by multidimensional NMR methods. The intermediate has partial native-like secondary structure and backbone topology, consistent with our earlier native state hydrogen exchange results. However, non-native hydrophobic interactions exist throughout the structure. These and other results in the literature suggest that non-native hydrophobic interactions may occur generally in partially folded states. This can alter the interpretation of mutational protein engineering results in terms of native-like side chain interactions. In addition, since the intermediate exists after the rate-limiting step and Rd-apocyt b(562) folds very rapidly (k(f) approximately 10(4) s(-1)), these results suggest that non-native hydrophobic interactions, in the absence of topological misfolding, are repaired too rapidly to slow folding and cause the accumulation of folding intermediates. More generally, these results illustrate an approach for determining the high-resolution structure of folding intermediates.     

Diversification of porcine MHC class II genes: evidence for selective advantage

The major histocompatibility complex (MHC) is an immunological gene-dense region of high diversity in mammalian species. Sus scrofa was domesticated by at least six independent events over Eurasia during the Holocene period. It has been hypothesized that the level and distribution of MHC variation in pig populations reflect genetic selection and environmental influences. In an effort to define the complexity of MHC polymorphisms and the role of selection in the generation of class II gene diversity (DQB, DRB1, and pseudogene ΨDRB3), DNA from globally distributed unrelated domestic pigs of European and Asian origins and a Suidae out-group was analyzed. The number of pseudogene alleles identified (ΨDRB3 33) was greater than those found in the expressed genes (DQB 20 and DRB1 23) but the level of observed heterozygosity (ΨDRB3 0.452, DQB 0.732, and DRB1 0.767) and sequence diversity (ΨDRB3 0.029, DQB 0.062, and DRB1 0.074) were significantly lower in the pseudogene, respectively. The substitution ratios reflected an excess of d _N (DQB 1.476, DRB1 1.724, and ΨDRB3 0.508) and the persistence of expressed gene alleles suggesting the influence of balancing selection, while the pseudogene was undergoing purifying selection. The lack of a clear MHC phylogeographic tree, coupled with close genetic distances observed between the European and Asian populations (DQB 0.047 and DRB1 0.063) suggested that unlike observations using mtDNA, the MHC diversity lacks phylogeographic structure and appears to be globally uniform. Taken together, these results suggest that, despite regional differences in selective breeding and environments, no skewing of MHC diversity has occurred. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Energetics and cooperativity of the hydrogen bonding and anchor interactions that bind peptides to MHC class II protein

The complexity of the interaction between major histocompatibility complex class II (MHC II) proteins and peptide ligands has been revealed through structural studies and crystallographic characterization. Peptides bind through side-chain "anchor" interactions with MHC II pockets and an extensive array of genetically conserved hydrogen bonds to the peptide backbone. Here we quantitatively investigate the kinetic hierarchy of these interactions. We present results detailing the impact of single side-chain mutations of peptide anchor residues on dissociation rates, utilizing two I-A(d)-restricted peptides, one of which has a known crystal structure, and 24 natural and non-natural amino acid mutant variants of these peptides. We find that the N-terminal P1, P4 and P6 anchor-pocket interactions can make significant contributions to binding stability. We also investigate the interactions of these peptides with four I-A(d) MHC II proteins, each mutated to disrupt conserved hydrogen bonds to the peptide backbone. These complexes exhibit kinetic behavior suggesting that binding energy is disproportionately invested near the peptide N terminus for backbone hydrogen bonds. We then evaluate the effects of simultaneously modifying both anchor and hydrogen bonding interactions. A quantitative analysis of 71 double mutant cycles reveals that there is little apparent cooperativity between anchor residue interactions and hydrogen bonds, even when they are directly adjacent (<5A).     

Fast folding of a ribozyme by stabilizing core interactions: evidence for multiple folding pathways in RNA

Folding of the Tetrahymena ribozyme under physiological conditions in vitro is limited by slow conversion of long-lived intermediates to the active structure. These intermediates arise because the most stable domain of the ribozyme folds 10-50 times more rapidly than the core region containing helix P3. Native gel electrophoresis and time-resolved X-ray-dependent hydroxyl radical cleavage revealed that mutations that weaken peripheral interactions between domains accelerated folding fivefold, while a point mutation that stabilizes P3 enabled 80 % of the mutant RNA to reach the native conformation within 30 seconds at 22 degrees C. The P3 mutation increased the folding rate of the catalytic core as much as 50-fold, so that both domains of the ribozyme were formed at approximately the same rate. The results show that the ribozyme folds rapidly without significantly populating metastable intermediates when native interactions in the ribozyme core are stabilized relative to peripheral structural elements.     

Trans-species polymorphism and evidence of selection on class II MHC loci in tuco-tucos (Rodentia: Ctenomyidae)

Balancing selection acting over the evolutionary history of a lineage can result in the retention of alleles among species for longer than expected under neutral evolution. The associated pattern of trans-species polymorphism, in which similar or even identical alleles are shared among species, is often used to infer that balancing selection has occurred. The genes of the major histocompatibility complex (MHC) are thought to be subject to balancing selection that maintains alleles associated with response to specific pathogens. To explore the role of balancing selection in shaping MHC diversity in ctenomyid rodents, we examined allelic variability at the class II DRB and DQA loci in 18 species in the genus Ctenomys. Previous studies of four of these species had revealed significant within-population evidence of positive selection on MHC loci. The current study expands upon these analyses to (1) evaluate among-species evidence of positive selection and (2) explore the potential for balancing selection on MHC genes. Interspecific nucleotide sequence variation revealed significant evidence of positive selection on the DRB and DQA loci. At the same time, comparisons of phylogenetic trees for these MHC loci with a putative species tree based on mitochondrial sequence data revealed multiple examples of trans-specific polymorphism, including sharing of identical DRB and DQA alleles among distantly related species of Ctenomys. These findings suggest that MHC genes in these animals have historically been subject to balancing selection and yield new insights into the complex suite of forces shaping MHC diversity in free-living vertebrates.     

Inhibition of EBV-induced lymphoproliferation by CD4(+) T cells specific for an MHC class II promiscuous epitope

Posttransplant lymphoproliferative disorder (PTLD) and B cell lymphomas induced by EBV continue to be a major life-threatening complication in transplant patients. The establishment and enhancement of T cell immunity to EBV before transplantation and immunosuppressive therapy could help diminish these complications, but the lack of an effective vaccine has limited this prophylactic approach. We describe here the identification of a peptide epitope from the EBV EBNA2 Ag that is capable of inducing in vitro CD4(+) T cell responses that inhibit the EBV-mediated B lymphocyte proliferation associated with PTLD. Most significantly, T cell responses to the EBNA2 epitope were found to be restricted by numerous MHC class II alleles (DR1, DR7, DR16, DR52, DQ2, and DQ7), indicating that this peptide is highly promiscuous and would be recognized by a large proportion (>50%) of the general population. These results are relevant for the design of a simple, inexpensive and widely applicable peptide-based vaccine to prevent PTLD in solid organ transplant patients.     

Regulation of MHC class II gene expression by the class II transactivator

MHC class II molecules are pivotal for the adaptive immune system, because they guide the development and activation of CD4+ T helper cells. Fulfilling these functions requires that the genes encoding MHC class II molecules are transcribed according to a strict cell-type-specific and quantitatively modulated pattern. This complex gene-expression profile is controlled almost exclusively by a single master regulatory factor, which is known as the class II transactivator. As we discuss here, differential activation of the three independent promoters that drive expression of the gene encoding the class II transactivator ultimately determines the exquisitely regulated pattern of MHC class II gene expression.     

Immunologic hierarchy,class II MHC promiscuity,and epitope spreading of a melanoma helper peptide vaccine

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4⁺ T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8⁺ T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund’s adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4⁺ and CD8⁺ T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4⁺ T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3_281–295 (49 %) and tyrosinase_386–406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8⁺ T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4⁺ and CD8⁺ T cell responses against melanoma antigens. CD4⁺ T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8⁺ T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.     

Towards a systems understanding of MHC class I and MHC class II antigen presentation

The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review.     

Xenopus MHC class II molecules. II. Polymorphism as determined by two-dimensional gel electrophoresis

The class II antigens from four inbred strains of Xenopus laevis (r, f, g, and j haplotypes) and six gynogenetic LG clones (two Xenopus laevis, two Xenopus gilli haplotypes) with functionally well-defined MHC types have been immunoprecipitated with the rabbit anti-human class II beta-chain serum anti-p29boost and analyzed by two-dimensional gel electrophoresis. The glycosylated material from 15-hr biosynthetically labeled cells runs as a broad fuzzy band around 33kD that, upon removal of N-linked glycans by Endo F, resolves into upper beta-chain bands and lower alpha-chain bands. Both the glycosylated and deglycosylated class II antigens give rise to multiple IEF spots in evenly spaced arrays (alpha-chain: two to eight spots in one to three arrays, beta-chain: two to 12 spots in one to five arrays). Both chains are polymorphic and both map to the functionally defined MHC. The large number of spots argues for multiple class II antigens; by radioactive N-terminal sequencing, two homologous alpha-chains and five beta-chains are present in the f haplotype. By comparison with MHC-linked alloantisera, anti-p29boost recognizes all major polymorphic class II molecules in Xenopus laevis. A selection of outbred animals were typed by using an IEF procedure requiring only a million PHA-stimulated blood cells.     

MHC class II proteins and disease: a structural perspective

MHC class II molecules on the surface of antigen-presenting cells display a range of peptides for recognition by the T-cell receptors of CD4+ T helper cells. Therefore, MHC class II molecules are central to effective adaptive immune responses, but conversely, genetic and epidemiological data have implicated these molecules in the pathogenesis of autoimmune diseases. Indeed, the strength of the associations between particular MHC class II alleles and disease render them the main genetic risk factors for autoimmune disorders such as type 1 diabetes. Here, we discuss the insights that the crystal structures of MHC class II molecules provide into the molecular mechanisms by which sequence polymorphisms might contribute to disease susceptibility.     

Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation

Class II major histocompatibility molecules are the primary susceptibility locus for many autoimmune disorders, including type 1 diabetes. Human DQ8 and I-A(g7), in the NOD mouse model of spontaneous autoimmune diabetes, confers diabetes risk by modulating presentation of specific islet peptides in the thymus and periphery. We used an in silico molecular docking program to screen a large "druglike" chemical library to define small molecules capable of occupying specific structural pockets along the I-A(g7) binding groove, with the objective of influencing presentation to T cells of the autoantigen insulin B chain peptide consisting of amino acids 9-23. In this study we show, using both murine and human cells, that small molecules can enhance or inhibit specific TCR signaling in the presence of cognate target peptides, based upon the structural pocket targeted. The influence of compounds on the TCR response was pocket dependent, with pocket 1 and 6 compounds inhibiting responses and molecules directed at pocket 9 enhancing responses to peptide. At nanomolar concentrations, the inhibitory molecules block the insulin B chain peptide consisting of amino acids 9-23, endogenous insulin, and islet-stimulated T cell responses. Glyphosine, a pocket 9 compound, enhances insulin peptide presentation to T cells at concentrations as low as 10 nM, upregulates IL-10 secretion, and prevents diabetes in NOD mice. These studies present a novel method for identifying small molecules capable of both stimulating and inhibiting T cell responses, with potentially therapeutic applications.     

Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC

CD4 is a coreceptor for binding of T cells to APC and the primary receptor for HIV. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cys130 in one monomer and Cys159 in the other one. Mild reduction of the extracellular part of CD4 resulted in formation of disulfide-linked dimers, which supports the domain-swapped model. The functional significance of dimer formation for coreceptor function was tested using cells expressing wild-type or disulfide-bond mutant CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function. Modeling of the complex of the TCR and domain-swapped CD4 dimer bound to class II MHC and Ag supports the domain-swapped dimer as the immune coreceptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation is also accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred coreceptor for binding to APC.