P1 Pocket Research Articles

ChemInform Abstract: Mechanisms of Macromolecular Protease Inhibitors

AbstractReview: 49 refs.

HLA-DM captures partially empty HLA-DR molecules for catalyzed removal of peptide

The mechanisms of HLA-DM catalyzed peptide exchange remain uncertain. We found that all stages of the interaction of DM with HLA-DR were dependent on the occupancy state of the peptide binding groove. High-affinity peptides were protected from removal by DM through two mechanisms: peptide binding induced dissociation of a long-lived complex of empty DR and DM, and high-affinity DR-peptide complexes bound DM only very slowly. Non-binding covalent DR-peptide complexes were converted to efficient DM binders upon truncation of an N-terminal peptide segment that emptied the P1 pocket and disrupted conserved hydrogen bonds to MHC. DM thus only binds to DR conformers in which a critical part of the binding site is vacant, due to spontaneous peptide motion.

Molecular and structural determinants of adamantyl susceptibility to HLA-DRs allelic variants: an in silico approach to understand the mechanism of MLEs

Class II major histocompatibility complex (MHC II) molecules as expressed by antigen-presenting cells are heterodimeric cell-surface glycoprotein receptors that are fundamental in initiating and propagating an immune response by presenting tumor-associated antigenic peptides to CD4(+)/T(H) cells. The loading efficiency of such peptides can be improved by small organic compounds (MHC Loading Enhancers-MLEs), that convert the non-receptive peptide conformation of MHC II to a peptide-receptive conformation. In a reversible reaction, these compounds open up the binding site of MHC II molecules by specific interactions with a yet undefined pocket. Here, we performed molecular docking and molecular dynamics simulation studies of adamantyl compounds on the predicted cavity around the P1 pocket of 2 allelic variants of HLA-DRs. The purpose was to investigate the suitability of adamantyl compounds as MLEs at the dimorphic β86 position. Docking studies revealed that besides numerous molecular interactions formed by the adamantyl compounds, Asnβ82, Tyrβ83, and Thrβ90 are the crucial amino acid residues that are characterized as the "sensors" of peptide loading. Molecular dynamics simulation studies exposed the dynamical structural changes that HLA-DRs adopted as a response to binding of 3-(1-adamantyl)-5-hydrazidocarbonyl-1H-pyrazole (AdCaPy). The conformations of AdCaPy complexed with the Glyβ86 HLA-DR allelic variant are well correlated with the stabilized form of peptide-loaded HLA-DRs, further confirming the role of AdCaPy as a MLE. Hydrogen bonding interaction analysis clearly demonstrated that after making suitable contacts with AdCaPy, HLA-DR changes its local conformation. However, AdCaPy complexed with HLA-DR having Valβ86 at the dimorphic position did not accommodate AdCaPy as MLE due to steric hindrance caused by the valine.

Diabetogenic T cells recognize insulin bound to IAg7in an unexpected, weakly binding register

A peptide derived from the insulin B chain contains a major epitope for diabetogenic CD4(+) T cells in the NOD mouse model of type 1 diabetes (T1D). This peptide can fill the binding groove of the NOD MHCII molecule, IA(g7), in a number of ways or "registers." We show here that a diverse set of NOD anti-insulin T cells all recognize this peptide bound in the same register. Surprisingly, this register results in the poorest binding of peptide to IA(g7). The poor binding is due to an incompatibility between the p9 amino acid of the peptide and the unique IA(g7) p9 pocket polymorphisms that are strongly associated with susceptibility to T1D. Our findings suggest that the association of autoimmunity with particular MHCII alleles may be do to poorer, rather than more favorable, binding of the critical self-epitopes, allowing T-cell escape from thymic deletion.

The diabetogenic mouse MHC class II molecule I-Ag7 is endowed with a switch that modulates TCR affinity

Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-Ag7, typically have a small, uncharged amino acid residue at position 57 of their beta chain (beta57); this results in the absence of a salt bridge between beta57 and Argalpha76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Argalpha76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3beta. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 A) electrostatic interactions existed among Argalpha76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue beta57 of the I-Ag7 and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.

Design, synthesis and evaluation of high-affinity binders for the celiac disease associated HLA-DQ2 molecule

Celiac disease is caused by uncontrolled CD4 T-cell responses directed to wheat-derived gluten peptides bound to the disease predisposing HLA-DQ molecules. The only available treatment is a life-long gluten-free diet which is complicated by the widespread use of wheat-derived gluten in the food industry. As the binding of gluten-derived peptides is a prerequisite for the induction of the inflammatory T-cell response, blockers that would prevent gluten peptide binding to the HLA-DQ molecules might be used as an alternative to the gluten-free diet. In the present study we have analyzed the binding properties of a set of previously identified natural ligands for HLA-DQ2, the primary disease predisposing allele. An in silico method, Epibase, ranked these peptides and the top one, a peptide with a nine amino acid core FVAEYEPVL, was measured among these peptides as the peptide with the highest binding affinity for HLA-DQ2. In a stepwise approach we subsequently tested the impact of N-terminal extensions and systematic single amino acid substitutions within the core of this peptide which revealed that an N-terminal extension with the tripeptide sequence ADA increased binding affinity 5- to 6-fold. In addition the substitution analysis indicated which amino acids were most preferred at anchor residues in the lead peptide, generally leading to an increase of binding affinity with a factor of 2. Next we tested which combinations of such preferred amino acids yielded the best results. The combined results indicate that a peptide with sequence ADAYDYESEELFAA (core in bold) had superior binding properties. This peptide was chosen as a lead peptide for further optimization with non-natural amino acids at the p1 position, since molecular modeling indicated that none of the natural amino acids is able to optimally occupy the p1 pocket. A set of 8 non-proteinogenic amino acids was designed, synthesized and incorporated in the lead peptide (and in two control peptides) and tested for binding to HLA-DQ2. The results indicate that the effect of the incorporation of these non-proteinogenic amino acids depended on the peptide in which they were incorporated and that the maximum increase in binding affinity obtained was approximately 2-fold. Altogether lead sequences were obtained that have a binding affinity for HLA-DQ2 that is 100- to 200-fold higher compared to that of the gluten-derived peptide that has the highest affinity for HLA-DQ2. Such peptides are candidate lead peptides for further optimization. Our results, however, also indicate that in order to obtain further significant increases in binding affinity alternative approaches will have to be explored.

Compounds Binding to the S2−S3 Pockets of Thrombin

A set of compounds designed to bind to the S2-S3 pockets of thrombin was prepared. These compounds included examples with no interactions in the S1 pocket. Proline, a common P2 in many thrombin inhibitors, was combined with known P3 residues and P1 substituents of varying size and lipophilicity. Binding constants were determined using surface plasmon resonance (SPR) biosensor technology and were found to be in good agreement with results from an enzyme assay. A dramatic increase in affinity (100-1000 times) was seen for compounds incorporating an amino group capable of forming a hydrogen bond with gly216 in the protein backbone. The ligand efficiency was increased by including substituents that form stronger hydrophobic interactions with the P1 pocket. The binding mode was confirmed by X-ray analysis, which revealed the anticipated binding motif that included hydrogen bonds as well as a tightly bound water molecule. A QSAR model indicated that hydrogen bonding and lipophilicity were important for the prediction of binding constants. The results described here may have implications for how directed compound libraries for shallow protein pockets, like S2 and S3 in serine proteases, can be designed.

Structure-based discovery of dengue virus protease inhibitors

Dengue virus belongs to the family Flaviviridae and is a major emerging pathogen for which the development of vaccines and antiviral therapy has seen little success. The NS3 viral protease is a potential target for antiviral drugs since it is required for virus replication. The goal of this study was to identify novel dengue virus (type 2; DEN2V) protease inhibitors for eventual development as effective anti-flaviviral drugs. The EUDOC docking program was used to computationally screen a small-molecule library for compounds that dock into the P1 pocket and the catalytic site of the DEN2V NS3 protease domain apo-structure [Murthy, K., Clum, S., Padmanabhan, R., 1999. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects. J. Biol. Chem. 274, 5573–5580] and the Bowman–Birk inhibitor-bound structure [Murthy, K., Judge, K., DeLucas, L., Padmanabhan, R., 2000. Crystal structure of dengue virus NS3 protease in complex with a Bowman–Birk inhibitor: implications for flaviviral polyprotein processing and drug design. J. Mol. Biol. 301, 759–767]. The top 20 computer-identified hits that demonstrated the most favorable scoring “energies” were selected for in vitro assessment of protease inhibition. Preliminary protease activity assays demonstrated that more than half of the tested compounds were soluble and exhibited in vitro inhibition of the DEN2V protease. Two of these compounds also inhibited viral replication in cell culture experiments, and thus are promising compounds for further development.

The role of HLA-DQ8 beta57 polymorphism in the anti-gluten T-cell response in coeliac disease.

Major histocompatibility complex (MHC) class II alleles HLA-DQ8 and the mouse homologue I-A(g7) lacking a canonical aspartic acid residue at position beta57 are associated with coeliac disease and type I diabetes. However, the role of this single polymorphism in disease initiation and progression remains poorly understood. The lack of Asp 57 creates a positively charged P9 pocket, which confers a preference for negatively charged peptides. Gluten lacks such peptides, but tissue transglutaminase (TG2) introduces negatively charged residues at defined positions into gluten T-cell epitopes by deamidating specific glutamine residues on the basis of their spacing to proline residues. The commonly accepted model, proposing that HLA-DQ8 simply favours binding of negatively charged peptides, does not take into account the fact that TG2 requires inflammation for activation and that T-cell responses against native gluten peptides are found, particularly in children. Here we show that beta57 polymorphism promotes the recruitment of T-cell receptors bearing a negative signature charge in the complementary determining region 3beta (CDR3beta) during the response against native gluten peptides presented by HLA-DQ8 in coeliac disease. These T cells showed a crossreactive and heteroclitic (stronger) response to deamidated gluten peptides. Furthermore, gluten peptide deamidation extended the T-cell-receptor repertoire by relieving the requirement for a charged residue in CDR3beta. Thus, the lack of a negative charge at position beta57 in MHC class II was met by negatively charged residues in the T-cell receptor or in the peptide, the combination of which might explain the role of HLA-DQ8 in amplifying the T-cell response against dietary gluten.

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: A molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.

Model for the Peptide-Free Conformation of Class II MHC Proteins

BackgroundMajor histocompatibility complex proteins are believed to undergo significant conformational changes concomitant with peptide binding, but structural characterization of these changes has remained elusive.Methodology/Principal FindingsHere we use molecular dynamics simulations and experimental probes of protein conformation to investigate the peptide-free state of class II MHC proteins. Upon computational removal of the bound peptide from HLA-DR1-peptide complex, the α50-59 region folded into the P1-P4 region of the peptide binding site, adopting the same conformation as a bound peptide. Strikingly, the structure of the hydrophobic P1 pocket is maintained by engagement of the side chain of Phe α54. In addition, conserved hydrogen bonds observed in crystal structures between the peptide backbone and numerous MHC side chains are maintained between the α51-55 region and the rest of the molecule. The model for the peptide-free conformation was evaluated using conformationally-sensitive antibody and superantigen probes predicted to show no change, moderate change, or dramatic changes in their interaction with peptide-free DR1 and peptide-loaded DR1. The binding observed for these probes is in agreement with the movements predicted by the model.Conclusion/SignificanceThis work presents a molecular model for peptide-free class II MHC proteins that can help to interpret the conformational changes known to occur within the protein during peptide binding and release, and can provide insight into possible mechanisms for DM action.

Anchor Side Chains of Short Peptide Fragments Trigger Ligand-Exchange of Class II MHC Molecules

Class II MHC molecules display peptides on the cell surface for the surveillance by CD4+ T cells. To ensure that these ligands accurately reflect the content of the intracellular MHC loading compartment, a complex processing pathway has evolved that delivers only stable peptide/MHC complexes to the surface. As additional safeguard, MHC molecules quickly acquire a ‘non-receptive’ state once they have lost their ligand. Here we show now that amino acid side chains of short peptides can bypass these safety mechanisms by triggering the reversible ligand-exchange. The catalytic activity of dipeptides such as Tyr-Arg was stereo-specific and could be enhanced by modifications addressing the conserved H-bond network near the P1 pocket of the MHC molecule. It affected both antigen-loading and ligand-release and strictly correlated with reported anchor preferences of P1, the specific target site for the catalytic side chain of the dipeptide. The effect was evident also in CD4+ T cell assays, where the allele-selective influence of the dipeptides translated into increased sensitivities of the antigen-specific immune response. Molecular dynamic calculations support the hypothesis that occupation of P1 prevents the ‘closure’ of the empty peptide binding site into the non-receptive state. During antigen-processing and -presentation P1 may therefore function as important “sensor” for peptide-load. While it regulates maturation and trafficking of the complex, on the cell surface, short protein fragments present in blood or lymph could utilize this mechanism to alter the ligand composition on antigen presenting cells in a catalytic way.

Fuzzy Tricentric Pharmacophore Fingerprints. 2. Application of Topological Fuzzy Pharmacophore Triplets in Quantitative Structure−Activity Relationships

Topological fuzzy pharmacophore triplets (2D-FPT), using the number of interposed bonds to measure separation between the atoms representing pharmacophore types, were employed to establish and validate quantitative structure-activity relationships (QSAR). Thirteen data sets for which state-of-the-art QSAR models were reported in literature were revisited in order to benchmark 2D-FPT biological activity-explaining propensities. Linear and nonlinear QSAR models were constructed for each compound series (following the original author's splitting into training/validation subsets) with three different 2D-FPT versions, using the genetic algorithm-driven Stochastic QSAR sampler (SQS) to pick relevant triplets and fit their coefficients. 2D-FPT QSARs are computationally cheap, interpretable, and perform well in benchmarking. In a majority of cases (10/13), default 2D-FPT models validated better than or as well as the best among those reported, including 3D overlay-dependent approaches. Most of the analogues series, either unaffected by protonation equilibria or unambiguously adopting expected protonation states, were equally well described by rule- or pKa-based pharmacophore flagging. Thermolysin inhibitors represent a notable exception: pKa-based flagging boosts model quality, although--surprisingly--not due to proteolytic equilibrium effects. The optimal degree of 2D-FPT fuzziness is compound set dependent. This work further confirmed the higher robustness of nonlinear over linear SQS models. In spite of the wealth of studied sets, benchmarking is nevertheless flawed by low intraset diversity: a whole series of thereby caused artifacts were evidenced, implicitly raising questions about the way QSAR studies are conducted nowadays. An in-depth investigation of thrombin inhibition models revealed that some of the selected triplets make sense (one of these stands for a topological pharmacophore covering the P1 and P2 binding pockets). Nevertheless, equations were either unable to predict the activity of the structurally different ligands or tended to indiscriminately predict any compound outside the training family to be active. 2D-FPT QSARs do however not depend on any common scaffold required for molecule superimposition and may in principle be trained on hand of diverse sets, which is a must in order to obtain widely applicable models. Adding (assumed) inactives of various families for training enabled discovery of models that specifically recognize the structurally different actives.

The relative energetic contributions of dominant P1 pocket versus hydrogen bonding interactions to peptide:class II stability: Implications for the mechanism of DM function

Peptides are bound to MHC class II molecules by an array of hydrogen bonds between conserved MHC class II protein side-chains and the peptide backbone and through interactions between MHC protein pockets and peptide side-chain anchors. The crystal structure of murine I-A k protein with peptide shows a network of electrostatic interactions with the P1 aspartic acid anchor and an arginine in the P1 pocket that are thought to constitute the major stabilizing interaction between peptide and MHC. In this paper, have explored the relative energetic contribution of this dominant P1 pocket interaction with that made by a genetically conserved hydrogen bond which is formed by the beta 81 histidine residue and the main chain of the bound peptide. We have performed peptide dissociation experiments using antigenic peptides or variants that have altered side-chain interactions with the I-A k P1 pocket using either native I-A k or I-A k proteins mutated to disrupt the N-terminal hydrogen bond. The results demonstrate that the N-terminal hydrogen bonds in I-A k complexes make highly significant energetic contributions to the kinetic stabilities comparable to or greater than the energetic contribution of highly favorable P1 pocket interactions. Hence, we conclude that the kinetic stability of MHC class II:peptide complexes critically depends on two quite distinct molecular interactions between peptide and MHC located at the peptide's amino terminus. We discuss these results in light of the proposed mechanism for DM function.

Exploring the P2 and P3 ligand binding features for Hepatitis C virus NS3 protease using some 3D QSAR techniques

Several three-dimensional quantitative structure–activity relationship (3D-QSAR) models have been constructed using the comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and Catalyst pharmacophore feature building programs for a series of 26 truncated ketoacid inhibitors designed particularly for exploring the P2 and P3 binding pockets of HCV NS3 protease. The structures of these inhibitors were built from a structure template extracted from the crystal structure of HCV NS3 protease. The structures were aligned through docking each inhibitor into the NS3 active site using program GOLD. The best CoMSIA model was identified from the stepwise analysis results and the corresponding pharmacophore features derived were used for constructing a pharmacophore hypothesis by the Catalyst program. Pharmacophore features obtained by CoMFA and CoMSIA are found to be in accord with each other and are both mapped onto the molecular 5 K surface of NS3 active site. These pharmacophore features were also compared with those obtained by the Catalyst program and mapped onto the same NS3 molecular surface. The pharmacophore building process was also performed for 20 boronic acid based NS3 inhibitors characterized by a long hydrophobic side chain attached at position P2. This latter pharmacophore hypothesis built by the Catalyst program was also mapped onto the molecular surface of NS3 active site to define a second hydrophobic feature at position P2. The possibility of using the pharmacophore features mapped P2 and P3 binding pocket to design more potent depeptidized NS3 inhibitors was discussed.

Conformation of MHC class II I-Ag7 is sensitive to the P9 anchor amino acid in bound peptide

Type I diabetes is a chronic autoimmune disease resulting in the destruction of insulin-producing beta cells in the pancreas. In humans, disease incidence is linked to expression of specific MHC class II alleles and in mice type I diabetes is associated with the class II allele I-A(g7). I-A(g7) contains a polymorphism that is shared by human class II alleles associated with the disease, at position 57 in the beta chain, in which aspartic acid is changed to a serine. The P9 pocket in the peptide-binding groove is in part shaped by beta57, and therefore the structure of this pocket is modified in I-A(g7). Using mAbs, we have previously determined that alternative conformations of I-A(g7) form in response to peptide binding. In this study, we have extended these findings by examining how peptides induce I-A(g7) molecules to adopt different conformations. By mutating the amino acid in the P9 position of either class II-associated invariant chain peptide (CLIP) or glutamic acid decarboxylase (GAD) 65 (207-220), we have determined that the chemical nature of the P9 anchor amino acid, either acidic or small hydrophobic, affects the overall conformation of the I-A(g7) class II molecule. T cell hybridomas specific for GAD 65 (207-220) in the context of I-A(g7) were also examined for recognition of I-A(g7) bound to GAD 65 (207-220), in which Glu(217) in the P9 position was changed to alanine. We found that although some TCRs were able to recognize both peptides in the context of I-A(g7), and thus both class II conformations, approximately one-third of the T cells tested were not able to recognize the alternate class II conformation formed with the mutated peptide. These results indicate that the I-A(g7) conformations may affect functional activation of T cells, and thus may play a role in autoimmunity.

Transamidation of Wheat Flour Inhibits the Response to Gliadin of Intestinal T Cells in Celiac Disease

Celiac disease is characterized by activation of HLA-DQ2/DQ8-restricted intestinal gluten-specific CD4(+) T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. Enzyme modifications of the immunodominant alpha-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Wheat flour was treated with microbial transglutaminase and lysine methyl ester; gliadin was subsequently extracted, digested, and deamidated. Gliadin-specific intestinal T-cell lines (iTCLs) were generated from biopsy specimens from 12 adult patients with celiac disease and challenged in vitro with different antigen preparations. Tissue transglutaminase-mediated transamidation with lysine or lysine methyl ester of p56-68 or gliadin in alkaline conditions inhibited the interferon gamma expression in iTCLs; also, binding to DQ2 was reduced but not abolished, as suggested by in silico analysis. Lysine methyl ester was particularly effective in abrogating the activity of gliadin. Notably, a block in the response was observed when iTCLs were challenged with gliadin extracted from flour pretreated with microbial transglutaminase and lysine methyl ester. Transamidation of wheat flour with a food-grade enzyme and an appropriate amine donor can be used to block the T cell-mediated gliadin activity. Considering the crucial role of adaptive immunity in celiac disease, our findings highlight the potential of the proposed treatment to prevent cereal toxicity.

Crystallographic Structure of the Human Leukocyte Antigen DRA, DRB3*0101: Models of a Directional Alloimmune Response and Autoimmunity

We describe structural studies of the human leukocyte antigen DR52a, HLA-DRA/DRB3*0101, in complex with an N-terminal human platelet integrin αII BβIII glycoprotein peptide which contains a Leu/Pro dimorphism. The 33:Leu dimorphism is the epitope for the T cell directed response in neonatal alloimmune thrombocytopenia and post-transfusion purpura in individuals with the αII BβIII 33:Pro allele, and defines the unidirectional alloimmune response. This condition is always associated with DR52a. The crystallographic structure has been refined to 2.25 Å. There are two αβ heterodimers to the asymmetric unit in space group P4 12 12. The molecule is characterized by two prominent hydrophobic pockets at either end of the peptide binding cleft and a deep, narrower and highly charged P4 opening underneath the beta 1 chain. Further, the peptide in the second molecule displays a sharp upward turn after pocket P9. The structure reveals the role of pockets and the distinctive basic P4 pocket, shared by DR52a and DR3, in selecting their respective binding peptide repertoire. We observe an interesting switch in a residue from the canonically assigned pocket 6 seen in prior class II structures to pocket 4. This occludes the P6 pocket helping to explain the distinctive “ 1–4–9” peptide binding motif. A β57 Asp → Val substitution abrogates the salt-bridge to α76 Arg and along with a hydrophobic β37 is important in shaping the P9 pocket. DRB3*0101 and DRB1*0301 belong to an ancestral haplotype and are associated with many autoimmune diseases linked to antigen presentation, but whereas DR3 is susceptible to type 1 diabetes DR52a is not. This dichotomy is explored for clues to the disease.

Small Organic Compounds Enhance Antigen Loading of Class II Major Histocompatibility Complex Proteins by Targeting the Polymorphic P1 Pocket

Major histocompatibility complex (MHC) molecules are a key element of the cellular immune response. Encoded by the MHC they are a family of highly polymorphic peptide receptors presenting peptide antigens for the surveillance by T cells. We have shown that certain organic compounds can amplify immune responses by catalyzing the peptide loading of human class II MHC molecules HLA-DR. Here we show now that they achieve this by interacting with a defined binding site of the HLA-DR peptide receptor. Screening of a compound library revealed a set of adamantane derivatives that strongly accelerated the peptide loading rate. The effect was evident only for an allelic subset and strictly correlated with the presence of glycine at the dimorphic position beta86 of the HLA-DR molecule. The residue forms the floor of the conserved pocket P1, located in the peptide binding site of MHC molecule. Apparently, transient occupation of this pocket by the organic compound stabilizes the peptide-receptive conformation permitting rapid antigen loading. This interaction appeared restricted to the larger Gly(beta86) pocket and allowed striking enhancements of T cell responses for antigens presented by these "adamantyl-susceptible" MHC molecules. As catalysts of antigen loading, compounds targeting P1 may be useful molecular tools to amplify the immune response. The observation, however, that the ligand repertoire can be affected through polymorphic sites form the outside may also imply that environmental factors could induce allergic or autoimmune reactions in an allele-selective manner.

Recombinant HLA-DP2 Binds Beryllium and Tolerizes Beryllium-Specific Pathogenic CD4+ T Cells

Chronic beryllium disease is a lung disorder caused by beryllium exposure in the workplace and is characterized by granulomatous inflammation and the accumulation of beryllium-specific, HLA-DP2-restricted CD4+ T lymphocytes in the lung that proliferate and secrete Th1-type cytokines. To characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we constructed rHLA-DP2 and rHLA-DP4 molecules consisting of the alpha-1 and beta-1 domains of the HLA-DP molecules genetically linked into single polypeptide chains. Peptide binding to rHLA-DP2 and rHLA-DP4 was consistent with previously published peptide-binding motifs for these MHC class II molecules, with peptide binding dominated by aromatic residues in the P1 pocket. 9Be nuclear magnetic resonance spectroscopy showed that beryllium binds to the HLA-DP2-derived molecule, with no binding to the HLA-DP4 molecule that differs from DP2 by four amino acid residues. Using beryllium-specific CD4+ T cell lines derived from the lungs of chronic beryllium disease patients, beryllium presentation to those cells was independent of Ag processing because fixed APCs were capable of presenting BeSO4 and inducing T cell proliferation. Exposure of beryllium-specific CD4+ T cells to BeSO4 -pulsed, plate-bound rHLA-DP2 molecules induced IFN-gamma secretion. In addition, pretreatment of beryllium-specific CD4+ T cells with BeSO4-pulsed, plate-bound HLA-DP2 blocked proliferation and IL-2 secretion upon re-exposure to beryllium presented by APCs. Thus, the rHLA-DP2 molecules described herein provide a template for engineering variants that retain the ability to tolerize pathogenic CD4+ T cells, but do so in the absence of the beryllium Ag.