P1 Pocket Research Articles

P012 Structure directed identification of human metabolite binding in the P4 pocket of MHC class-II DRB1∗15:01 and DRB1∗15:03 and implications for multiple sclerosis risk

Aim Robust models of HLA mediated drug hypersensitivity demonstrate that binding of small molecules in the peptide-binding groove may result in changes in conformation or register shift of bound peptide, leading to an altered immune response. We performed virtual screening of the complete Human Metabolite Database (HMDB) for docking to the multiple sclerosis (MS) susceptible DRB1 ∗ 15:01 allele and compared the results to the closely related yet non-susceptible DRB1 ∗ 15:03 allele; and assessed the potential impact on binding of human myelin basic peptide (MBP). Methods The crystal structure of DRB1∗15:01 was obtained from the protein data bank under accession number 1BX2. An in silico DRB1 ∗ 15:03 structure was generated from the known structure of DRB1 ∗ 15:01 by mutating residue Tyr 30 > His 30 . The target proteins ( DRB1 ∗ 15:01 and DRB1 ∗ 15:03 ) were prepared with N-acetyl-D-glucosamine (NAG) for docking with human metabolites using UCSF Chimera (v1.11.2). We conducted independent screenings to probe each pocket of the peptide binding groove for both proteins using HMDB library v3.6, which consists of 41993 human metabolites. Results We observed higher energy scores for metabolite binding to DRB1 ∗ 15:01 than DRB1 ∗ 15:03. We found that 78 and 82 metabolites bind strictly to DRB1 ∗ 15:01 or DRB1 ∗ 15:03 respectively, while 79 metabolites bind to both structures. Structural comparison of docked metabolites with DRB1 ∗ 15:01 and DRB1 ∗ 15:03 complexed with MBP revealed that Phenylalanine MBP 92 allows binding of metabolites in the P4 pocket of DRB1 ∗ 15:01 but Valine MBP 89 abrogates metabolite binding in the P1 pocket. We observed differences in the energy score for the binding of the same metabolites in the P4 pockets of DRB1 ∗ 15:01 and DRB1 ∗ 15:03 or different metabolites in the P4 pockets of DRB1 ∗ 15:01 and DRB1 ∗ 15:03 suggesting stronger binding to DRB1 ∗ 15:01. Further, several of these metabolites have been previously associated with MS. Conclusions Our study confirmed that specific, disease-associated human metabolites bind effectively with the most polymorphic P4 pockets of DRB1 ∗ 15:01, the primary MS susceptible allele in most populations. Our results suggest that endogenous human metabolites bound in specific pockets of HLA may be immunomodulatory and implicated in autoimmune disease.

Molecular basis for increased susceptibility of Indigenous North Americans to seropositive rheumatoid arthritis

ObjectiveThe pathogenetic mechanisms by which HLA-DRB1 alleles are associated with anticitrullinated peptide antibody (ACPA)-positive rheumatoid arthritis (RA) are incompletely understood. RA high-risk HLA-DRB1 alleles are known to share a common...

Unraveling the structural basis for the unusually rich association of human leukocyte antigen DQ2.5 with class-II-associated invariant chain peptides

Human leukocyte antigen (HLA)-DQ2.5 (DQA1*05/DQB1*02) is a class-II major histocompatibility complex protein associated with both type 1 diabetes and celiac disease. One unusual feature of DQ2.5 is its high class-II-associated invariant chain peptide (CLIP) content. Moreover, HLA-DQ2.5 preferentially binds the non-canonical CLIP2 over the canonical CLIP1. To better understand the structural basis of HLA-DQ2.5's unusual CLIP association characteristics, better insight into the HLA-DQ2.5·CLIP complex structures is required. To this end, we determined the X-ray crystal structure of the HLA-DQ2.5· CLIP1 and HLA-DQ2.5·CLIP2 complexes at 2.73 and 2.20 Å, respectively. We found that HLA-DQ2.5 has an unusually large P4 pocket and a positively charged peptide-binding groove that together promote preferential binding of CLIP2 over CLIP1. An α9-α22-α24-α31-β86-β90 hydrogen bond network located at the bottom of the peptide-binding groove, spanning from the P1 to P4 pockets, renders the residues in this region relatively immobile. This hydrogen bond network, along with a deletion mutation at α53, may lead to HLA-DM insensitivity in HLA-DQ2.5. A molecular dynamics simulation experiment reported here and recent biochemical studies by others support this hypothesis. The diminished HLA-DM sensitivity is the likely reason for the CLIP-rich phenotype of HLA-DQ2.5.

Fragment-Based Design, Synthesis, and Biological Evaluation of 1-Substituted-indole-2-carboxylic Acids as Selective Mcl-1 Inhibitors.

Based on a known selective Mcl-1 inhibitor, 6-chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1H-indole-2-carboxylic acid, we applied a fragment-based approach to obtain new molecules that extended into the p1 pocket of the BH3 groove and then exhibited binding selectivity for the Mcl-1 over the Bcl-2 protein. After we deconstructed the 1H-indole-2-carboxylic acid from the parental molecule, a benzenesulfonyl was substituted at the 1-position to adopt a geometry preferred for accessing the p1 pocket according to the binding mode of the parental molecule identified by X-ray crystallography. A linear relationship between the free energy of ligand binding (ΔG) and the count of non-hydrogen heavy atoms (HAC) was maintained during the molecular growing to occupy the p1 pocket. Finally, we not only obtained compound 12 with a 7.5-fold selectivity to Mcl-1 (Ki = 0.48 µM by fluorescence polarization) over Bcl-2 (Ki = 3.6 µM), but also provided evidence that additional occupation of the p1 pocket is more favorable for Mcl-1 than for Bcl-2 binding, and contributes more to Mcl-1 inhibition than occupation of the p2 pocket. Compound 12 exhibited a selective killing ability on Mcl-1-dependent cancer cells.

Design, synthesis, and activity evaluation of selective inhibitors of anti-apoptotic Bcl-2 proteins: The effects on the selectivity of the P1 pockets in the active sites

The anti-apoptotic Bcl-2 proteins are attractive targets for anti-cancer drug development, and the discovery of their selective inhibitors has become a research focus. In this Letter, obvious differences in the P1 pocket of the active site between Bcl-2, Bcl-xL, and Mcl-1 proteins were proposed by the structural comparison of these proteins. As a result, the groups in their inhibitors binding to the P1 pockets may have significant effect on the selectivity for these proteins. Based on this hypothesis, five types of derivatives of the lead compound B-1 were designed, and several highly selective inhibitors of Bcl-xL (E-1) or Mcl-1 proteins (G) were found. The selective inhibitors of Mcl-1 protein found in this Letter provide new structural types for the development of novel antitumor agents.

P049 Human metabolites bind to the P4 pocket of HLA-DRB1∗15:01, with implications for multiple sclerosis susceptibility

Aim Models of HLA mediated drug hypersensitivity suggest that binding of small molecules in the peptide binding groove results in changes in conformation of the bound peptide, leading to an aberrant immune response. We hypothesize that naturally occurring small molecules may similarly alter T-cell response in Multiple Sclerosis (MS), where susceptibility is strongly associated with DRB1 ∗ 15:01. We performed virtual screening of the complete Human Metabolite Database (HMDB) for docking in DRB1 ∗ 15:01 and assessed the potential impact on binding of human myelin basic peptide (MBP). Methods Virtual Screening for HLA-DRB1 ∗ 15:01 was conducted on a structure obtained in molecular dynamics simulations of the complex with bound MBP. Valine 89 and phenylalanine 92 of the MBP were identified as P1 and P4 anchor residues. Two independent screenings were conducted to probe pockets P1 and P4 in the structure of DRB1 ∗ 15:01 obtained from the protein data bank (PDB code 1BX2) using the HMDB library (v3.6), which consists of 41993 human metabolites. The virtual screening was conducted with UCSF Chimera v1.10.2, AUTODOCK v4.2, Clipper 1.6.0, Dockblaster 1.6.0 program. Results We observed the binding of metabolites specifically in the P4 pockets of HLA-DRB1 ∗ 15:01. We calculated energy score, vander-waals component, electrostatic component, polar solvation component, molecular charge, heavy atoms, polar contacts (3.3 A), nonpolar contacts (3.3–4.5 A), and number of conformations for the docked DRB1 ∗ 15:01 and metabolite complex. We used Dockblaster (v1.6.0) to select top ranked metabolites and investigate the distribution of their scores. On this basis, we obtained 361 significant metabolites from the docking results in the P4 pocket and ranked them on the basis of their energy score. Further, we found that Phenylalanine MBP92 allows the binding of metabolites in the P4 pocket of DRB1 ∗ 1501 but not Valine MBP of P1 pockets. Conclusions These results suggest that the binding of naturally occurring human metabolites in the P4 pockets of HLA-DRB1 ∗ 15:01 may subtly alters the TCR recognition site of HLA/MBP, possibly by changing the conformation in the normally bound position, or causing a register shift in the bound peptide. This in turn may bring about profound alterations in T-cell responsiveness, resulting in autoimmunity.

Abstract 4331: Discovery of potent 2-Indole-acylsulfonamide Mcl-1 inhibitors using structure guided fragment-based methods

Abstract Mcl-1 (Myeloid cell leukemia 1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and is one of the major acquired resistance mechanisms for many anticancer therapies. Targeting Mcl-1 with small molecules is very challenging because Mcl-1 exerts its effects through protein-protein interactions. Here we describe our continuing efforts in the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. We previously described the discovery of small-molecule Mcl-1 inhibitors containing a 2-indole-carboxylic core that primarily bind to the P2 pocket. In order to enhance potency, we extend our molecules to occupy the entire BH3 binding interface. A number of P4 site hits were identified using a fragment-based screening while the P2 pocket of Mcl-1 was saturated with our previously reported lead. P4 site ligands were then linked to our initial lead guided by the ternary Mcl-1 structure containing both P2 and P4 units using an acylsulfonamide as a synthetic handle that retained the critical charged-charged interaction with R263. X-ray co-crystal structures of new inhibitors occupying both P2 and P4 sites bound Mcl-1 provided detailed information on how these small-molecules bind to the target and guided subsequent potency optimization. From the efforts, we discovered a novel series of 2-indole-acylsulfonamide containing Mcl-1 inhibitors that exhibit low nanomolar binding affinities to the target and greater than 500-fold selectivity over Bcl-xL. These compounds represent useful starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers. Citation Format: Subrata Shaw, James Chris Tarr, Nicholas Pelz, Zhiguo Bian, Johannes Belmar, Bin Zhao, Craig Goodwin, Allison Arnold, John Sensintaffar, Olivia Rossanese, Taekyu Lee, Edward Olejniczak, Stephen Fesik. Discovery of potent 2-Indole-acylsulfonamide Mcl-1 inhibitors using structure guided fragment-based methods. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4331.

Design and structure–activity relationships of novel inhibitors of human rhinovirus 3C protease

Human rhinovirus (HRV) is a primary cause of common cold and is linked to exacerbation of underlying respiratory diseases such as asthma and COPD. HRV 3C protease, which is responsible for cleavage of viral polyprotein in to proteins essential for viral life-cycle, represents an important target. We have designed proline- and azetidine-based analogues of Rupintrivir that target the P2 pocket of the binding site. Potency optimization, aided with X-ray crystallography and quantum mechanical calculations, led to compounds with activity against a broad spectrum of HRV serotypes. Altogether, these compounds represent alternative starting points to identify promising leads in our continual efforts to treat HRV infections.

Structure-based design of N-substituted 1-hydroxy-4-sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncoprotein

Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-molecule inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 pocket of the protein. Significantly, target molecules were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort. Molecular modeling using the Site-Identification by Ligand Competitive Saturation (SILCS) approach was used to qualitatively direct ligand design as well as develop quantitative models for inhibitor binding affinity to Mcl-1 and the Bcl-2 relative Bcl-xL as well as for the specificity of binding to the two proteins. Results indicated hydrophobic interactions in the p2 pocket dominated affinity of the most favourable binding ligand (3bl: Ki = 31 nM). Compounds were up to 19-fold selective for Mcl-1 over Bcl-xL. Selectivity of the inhibitors was driven by interactions with the deeper p2 pocket in Mcl-1 versus Bcl-xL. The SILCS-based SAR of the present compounds represents the foundation for the development of Mcl-1 specific inhibitors with the potential to treat a wide range of solid tumours and hematological cancers, including acute myeloid leukemia.

Structural Re-engineering of the α-Helix Mimetic JY-1-106 into Small Molecules: Disruption of the Mcl-1-Bak-BH3 Protein-Protein Interaction with 2,6-Di-Substituted Nicotinates.

The disruption of aberrant protein-protein interactions (PPIs) with synthetic agents remains a challenging goal in contemporary medicinal chemistry but some progress has been made. One such dysregulated PPI is that between the anti-apoptotic Bcl-2 proteins, including myeloid cell leukemia-1 (Mcl-1), and the α-helical Bcl-2 homology-3 (BH3) domains of its pro-apoptotic counterparts, such as Bak. Herein, we describe the discovery of small-molecule inhibitors of the Mcl-1 oncoprotein based on a novel chemotype. Particularly, re-engineering of our α-helix mimetic JY-1-106 into 2,6-di-substituted nicotinates afforded inhibitors of comparable potencies but with significantly decreased molecular weights. The most potent inhibitor 2-(benzyloxy)-6-(4-chloro-3,5-dimethylphenoxy)nicotinic acid (1 r: Ki =2.90 μm) likely binds in the p2 pocket of Mcl-1 and engages R263 in a salt bridge through its carboxylic acid, as supported by 2D (1) H-(15) N HSQC NMR data. Significantly, inhibitors were easily accessed in just four steps, which will facilitate future optimization efforts.

Peptide motif analysis predicts alphaviruses as triggers for rheumatoid arthritis.

Rheumatoid arthritis (RA) develops in response to both genetic and environmental factors. The strongest genetic determinant is HLA-DR, where polymorphisms within the P4 and P6 binding pockets confer elevated risk. However, low disease concordance across monozygotic twin pairs underscores the importance of an environmental factor, probably infectious. The goal of this investigation was to predict the microorganism most likely to interact with HLA-DR to trigger RA under the molecular mimicry hypothesis. A set of 185 structural proteins from viruses or intracellular bacteria was scanned for regions of sequence homology with a collagen peptide that binds preferentially to DR4; candidates were then evaluated against a motif required for T cell cross-reactivity. The plausibility of the predicted agent was evaluated by comparison of microbial prevalence patterns to epidemiological characteristics of RA. Peptides from alphavirus capsid proteins provided the closest fit. Variations in the P6 position suggest that the HLA binding preference may vary by species, with Ross River virus, Chikungunya virus, and Mayaro virus peptides binding preferentially to DR4, and peptides from Sindbis/Ockelbo virus showing stronger affinity to DR1. The predicted HLA preference is supported by epidemiological studies of post-infection chronic arthralgia. Parallels between the cytokine profiles of RA and chronic alphavirus infection are discussed.

Obatoclax is a direct and potent antagonist of membrane-restricted Mcl-1 and is synthetic lethal with treatment that induces Bim.

BackgroundObatoclax is a clinical stage drug candidate that has been proposed to target and inhibit prosurvival members of the Bcl-2 family, and thereby contribute to cancer cell lethality. The insolubility of this compound, however, has precluded the use of many classical drug-target interaction assays for its study. Thus, a direct demonstration of the proposed mechanism of action, and preferences for individual Bcl-2 family members, remain to be established.MethodsEmploying modified proteins and lipids, we recapitulated the constitutive association and topology of mitochondrial outer membrane Mcl-1 and Bak in synthetic large unilamellar liposomes, and measured bakdependent bilayer permeability. Additionally, cellular and tumor models, dependent on Mcl-1 for survival, were employed.ResultsWe show that regulation of bilayer permeabilization by the tBid – Mcl-1 - Bak axis closely resemblesthe tBid - Bcl-XL - Bax model. Obatoclax rapidly and completely partitioned into liposomal lipid but also rapidly exchanged between liposome particles. In this system, obatoclax was found to be a direct and potent antagonist of liposome-bound Mcl-1 but not of liposome-bound Bcl-XL, and did not directly influence Bak. A 2.5 molar excess of obatoclax relative to Mcl-1 overcame Mcl-1-mediated inhibition of tBid-Bak activation. Similar results were found for induction of Bak oligomers by Bim. Obatoclax exhibited potent lethality in a cellmodel dependent on Mcl-1 for viability but not in cells dependent on Bcl-XL. Molecular modeling predicts that the 3-methoxy moiety of obatoclax penetrates into the P2 pocket of the BH3 binding site of Mcl-1. A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1. Systemic treatment of mice bearing Tsc2+/- Em-myc lymphomas (whose cells depend on Mcl-1 for survival) with obatoclax conferred a survival advantage compared to vehicle alone (median 31 days vs 22 days, respectively; p=0.003). In an Akt-lymphoma mouse model, the anti-tumor effects of obatoclax synergized with doxorubicin. Finally, treatment of the multiple myeloma KMS11 cell model (dependent on Mcl-1 for survival) with dexamethasone induced Bim and Bim-dependent lethality. As predicted for an Mcl-1 antagonist, obatoclax and dexamethasone were synergistic in this model.ConclusionsTaken together, these findings indicate that obatoclax is a potent antagonist of membranerestricted Mcl-1. Obatoclax represents an attractive chemical series to generate second generation Mcl-1 inhibitors.

Additive and interaction effects at three amino acid positions in HLA-DQ and HLA-DR molecules drive type 1 diabetes risk.

Variation in the human leukocyte antigen (HLA) genes accounts for one-half of the genetic risk in type 1 diabetes (T1D). Amino acid changes in the HLA-DR and HLA-DQ molecules mediate most of the risk, but extensive linkage disequilibrium complicates the localization of independent effects. Using 18,832 case-control samples, we localized the signal to 3 amino acid positions in HLA-DQ and HLA-DR. HLA-DQβ1 position 57 (previously known; P = 1 × 10(-1,355)) by itself explained 15.2% of the total phenotypic variance. Independent effects at HLA-DRβ1 positions 13 (P = 1 × 10(-721)) and 71 (P = 1 × 10(-95)) increased the proportion of variance explained to 26.9%. The three positions together explained 90% of the phenotypic variance in the HLA-DRB1-HLA-DQA1-HLA-DQB1 locus. Additionally, we observed significant interactions for 11 of 21 pairs of common HLA-DRB1-HLA-DQA1-HLA-DQB1 haplotypes (P = 1.6 × 10(-64)). HLA-DRβ1 positions 13 and 71 implicate the P4 pocket in the antigen-binding groove, thus pointing to another critical protein structure for T1D risk, in addition to the HLA-DQ P9 pocket.

Positional identification of RT1-B (HLA-DQ) as susceptibility locus for autoimmune arthritis.

Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.

New insights into stop codon recognition by eRF1.

In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be important for termination efficiency, but the recognition mechanism remains unclear. We characterized a region of the eRF1 N-terminal domain, the P1 pocket, that we had previously shown to be involved in termination efficiency. We performed alanine scanning mutagenesis of this region, and we quantified in vivo readthrough efficiency for each alanine mutant. We identified two residues, arginine 65 and lysine 109, as critical for recognition of the three stop codons. We also demonstrated a role for the serine 33 and serine 70 residues in UGA decoding in vivo. NMR analysis of the alanine mutants revealed that the correct conformation of this region was controlled by the YxCxxxF motif. By combining our genetic data with a structural analysis of eRF1 mutants, we were able to formulate a new model in which the stop codon interacts with eRF1 through the P1 pocket.

Design, synthesis and structure–activity relationship studies of morpholino-1H-phenalene derivatives that antagonize Mcl-1/Bcl-2

We report herein characteristic studies of Mcl-1 and Bcl-2 dual inhibitors. It was found that a protruding carbonyl group forming hydrogen bond with R263 plays a predominant role compared with the hydrophobic group that occupies the p2 pocket. A series of dual inhibitors representing different parts of the morpholino-1H-phenalene were designed, synthesized and evaluated.

Structure-based drug design of diphenyl α-aminoalkylphosphonates as prostate-specific antigen antagonists.

Here, we describe the mechanism of diphenyl α-aminoalkylphosphonate ester derivatives as potent inhibitors of prostate-specific antigen (PSA), a likely protease responsible for the advancement of prostate tumor progression. The AutoDock 4.2 molecular docking suite was utilized to model covalent and noncovalent binding of this class of inhibitors to predict crystallographic poses and compare experimental IC50 dose-response curves and in silico potencies for providing future more specific rational drug design. The new lead compound R/S-diphenyl[N-benzyloxycarbonylamino(4-carbamoylphenyl)methyl]phosphonate is being reported in this study as a potent inhibitor of PSA activity (IC50 = 250 nM; AutoDock Score = -8.29/-9.14 kJ·mol(-1) for R/S). Molecular dynamics (MD) simulations using GROMACS 4.6.5 was used to obtain trajectories of the top ligand and validate key interactions in the binding complex. A hydrogen-bonding map was used to confirm interactions between the lead compound and residues THR190, SER217, and SER227 in the P1 pocket. The modeling study introduces novel aminoalkylphosphonates as a potential drug candidate for targeting PSA by optimizing P1 binding affinities.

A molecular basis for citrullination dependent rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease, characterized by inflammation of synovial tissue, joint pannus and bone erosion. The human leukocyte antigen (HLA) locus plays a vital role in immunity, encoding highly polymorphic molecules that present peptides to T cell lymphocytes. RA has a strong association with a region of the HLA-DRB1 locus known as the `shared epitope' (SE) and the presence of autoantibodies specific for citrullinated proteins. The SE maps to a highly polymorphic N-terminal region of the HLA-DRβ chain around amino acids 70-74. This region encodes a positively charged residue at position 71 that is thought to dictate the amino acid that is accommodated in the P4 pocket of the antigen-binding groove. Citrullination, the conversion of positively charged arginine to polar citrulline, is a physiological process catalyzed by peptidyl arginine deiminase. Previous studies have shown that citrullination of self-antigens can significantly increase the affinity of epitopes for SE alleles. Here we provide a molecular basis for how citrullinated vimentin and aggrecan epitopes can be presented by the SE alleles, HLA-DRB1*0401 and HLA-DRB1*0404. Citrulline was accommodated in the electropositive P4 pocket of HLA-DRB1*0401/04, whilst arginine was not. In addition, the RA resistant HLA-DRB1*0402 allomorph was capable of binding both arginine and citrulline in its electronegative P4 pocket. Peptide elution studies revealed that arginine was presented by HLA-DRB1*0402 but not by HLA-DRB1*0401/04. Moreover, citrullinated vimentin showed a greater sensitivity to proteolysis by cathepsin L, when compared to unmodified vimentin, indicating that citrullination can impact the repertoire of self-antigens presented. Using HLA Class II tetramers, we identified citrullinated vimentin and aggrecan specific CD4+ T cells from both HLA-DRB1*0401+ RA patients and healthy controls. In RA patients, the number of autoreactive T cells correlated with disease activity and were deficient in regulatory T cells compared to healthy controls. Together these findings provide significant insight into the role citrullination plays in the pathogenesis of RA[1].

Susceptibility to HLA-DM Protein Is Determined by a Dynamic Conformation of Major Histocompatibility Complex Class II Molecule Bound with Peptide

HLA-DM mediates the exchange of peptides loaded onto MHCII molecules during antigen presentation by a mechanism that remains unclear and controversial. Here, we investigated the sequence and structural determinants of HLA-DM interaction. Peptides interacting nonoptimally in the P1 pocket exhibited low MHCII binding affinity and kinetic instability and were highly susceptible to HLA-DM-mediated peptide exchange. These changes were accompanied by conformational alterations detected by surface plasmon resonance, SDS resistance assay, antibody binding assay, gel filtration, dynamic light scattering, small angle x-ray scattering, and NMR spectroscopy. Surprisingly, all of those changes could be reversed by substitution of the P9 pocket anchor residue. Moreover, MHCII mutations outside the P1 pocket and the HLA-DM interaction site increased HLA-DM susceptibility. These results indicate that a dynamic MHCII conformational determinant rather than P1 pocket occupancy is the key factor determining susceptibility to HLA-DM-mediated peptide exchange and provide a molecular mechanism for HLA-DM to efficiently target unstable MHCII-peptide complexes for editing and exchange those for more stable ones.

Structural Basis for the Specific Recognition of the Major Antigenic Peptide from the Japanese Cedar Pollen Allergen Cry j 1 by HLA-DP5

The major allergen, Cry j 1, was isolated from Japanese cedar Cryptomeria japonica (Cry j) pollen and was shown to react with immunoglobulin E antibodies in the sera from pollinosis patients. We previously reported that the frequency of HLA-DP5 was significantly higher in pollinosis patients and the immunodominant peptides from Cry j 1 bound to HLA-DP5 to activate Th2 cells. In the present study, we determined the crystal structure of the HLA-DP5 heterodimer in complex with a Cry j 1-derived nine-residue peptide, at 2.4Å resolution. The peptide-binding groove recognizes the minimal peptide with 10 hydrogen bonds, including those between the negatively charged P1 pocket and the Lys side chain at the first position in the peptide sequence. We confirmed that HLA-DP5 exhibits the same Cry j 1-binding mode in solution, through pull-down experiments using structure-based mutations of Cry j 1. We also identified the characteristic residues of HLA-DP5 that are responsible for the distinct properties of the groove, by comparing the structure of HLA-DP5 and the previously reported structures of HLA-DP2 in complexes with pDRA of the self-antigen. The comparison revealed that the HLA-DP5·pCry j 1 complex forms several hydrogen bond/salt bridge networks between the receptor and the antigen that were not observed in the HLA-DP2·pDRA complex. Evolutionary considerations have led us to conclude that HLA-DP5 and HLA-DP2 represent two major groups of the HLA-DP family, in which the properties of the P1 and P4 pockets have evolved and acquired the present ranges of epitope peptide-binding specificities.