Crystal structure of the HLA-DM-HLA-DR1 complex defines mechanisms for rapid peptide selection.

HLA-DR molecules bind microbial peptides in an endosomal compartment and present them on the cell surface for CD4 T cell surveillance. HLA-DM plays a critical role in the endosomal peptide selection process. The structure of the HLA-DM-HLA-DR complex shows major rearrangements of the HLA-DR peptide-binding groove. Flipping of a tryptophan away from the HLA-DR1 P1 pocket enables major conformational changes that position hydrophobic HLA-DR residues into the P1 pocket. These conformational changes accelerate peptide dissociation and stabilize the empty HLA-DR peptide-binding groove. Initially, incoming peptides have access to only part of the HLA-DR groove and need to compete with HLA-DR residues for access to the P2 site and the hydrophobic P1 pocket. This energetic barrier creates a rapid and stringent selection process for the highest-affinity binders. Insertion of peptide residues into the P2 and P1 sites reverses the conformational changes, terminating selection through DM dissociation.

In vivo discovery of immunotherapy targets in the tumour microenvironment.

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments.

Mechanisms of peptide repertoire selection by HLA-DM.

Recently, crystal structures of key complexes in antigen presentation have been reported. HLA-DM functions in antigen presentation by catalyzing dissociation of an invariant chain remnant from the peptide binding groove and stabilizing empty MHC class II proteins in a peptide-receptive conformation. The crystal structure of a MHC class II-HLA-DM complex explains how HLA-DM stabilizes an otherwise short-lived transition state and promotes a rapid peptide exchange process that favors the highest-affinity ligands. HLA-DO has sequence similarity with MHC class II molecules yet inhibits antigen presentation. The structure of the HLA-DO-HLA-DM complex shows that it blocks HLA-DM activity as a substrate mimic. Alterations in the efficiency of DM-mediated peptide selection may contribute to autoimmune pathologies, which will be an exciting area for future investigation.

The macrophage mannose receptor promotes uptake of ADAMTS13 by dendritic cells.

ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by cleaving ultra-large VWF multimers on the surfaces of endothelial cells. Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura. The formation of autoantibodies depends on the activation of CD4(+) T cells. This process requires immune recognition, endocytosis, and subsequent processing of ADAMTS13 into peptides that are presented on MHC class II molecules to CD4(+) T cells by dendritic cells (DCs). In the present study, we investigated endocytosis of recombinant ADAMTS13 by immature monocyte-derived DCs using flow cytometry and confocal microscopy. After incubation of fluorescently labeled ADAMTS13 with DCs, significant uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked by the addition of EGTA and mannan. ADAMTS13 endocytosis was decreased in the presence of a blocking mAb directed toward the macrophage mannose receptor (MR). Furthermore, siRNA silencing of MR reduced the uptake of ADAMTS13 by DCs. In addition, in vitro binding studies confirmed the interaction of ADAMTS13 with the carbohydrate recognition domains of MR. The results of the present study indicate that sugar moieties on ADAMTS13 interact with MR, thereby promoting its endocytosis by APCs.

An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF.

In the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP), antibodies are directed toward the spacer domain of ADAMTS13. We have previously shown that region Y658-Y665 is involved. We now show that replacement of R660, Y661, or Y665 with alanine in ADAMTS13 reduced/abolished the binding of 2 previously isolated human monoclonal antibodies and polyclonal antibodies derived from plasma of 6 patients with acquired TTP. We investigated whether these residues also influenced cleavage of short von Willebrand factor (VWF) fragment substrate VWF115. An ADAMTS13 variant (R660A/Y661A/Y665A, ADAMTS13-RYY) showed a 12-fold reduced catalytic efficiency (k(cat)/K(m)) arising from greatly reduced (> 25-fold) binding, demonstrated by surface plasmon resonance. The influence of these residue changes on full-length VWF was determined with denaturing and flow assays. ADAMTS13-RYY had reduced activity in both, with proteolysis of VWF unaffected by autoantibody. Binding of ADAMTS13-RYY mutant to VWF was, however, similar to normal. Our results demonstrate that residues within Y658-Y665 of the ADAMTS13 spacer domain that are targeted by autoantibodies in TTP directly interact with a complementary exosite (E1660-R1668) within the VWF A2 domain. Residues R660, Y661, and Y665 are critical for proteolysis of short VWF substrates, but wider domain interactions also make important contributions to cleavage of full-length VWF.

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain.

BACKGROUND: The majority of patients diagnosed with thrombotic thrombocytopenic purpura have autoantibodies directed towards the spacer domain of ADAMTS13. DESIGN AND METHODS: In this study we explored the epitope specificity and immunoglobulin class and immunoglobulin G subclass distribution of anti-ADAMTS13 antibodies. The epitope specificity of anti-spacer domain antibodies was examined using plasma from 48 patients with acute acquired thrombotic thrombocytopenic purpura by means of immunoprecipitation of ADAMTS13 variants containing single or multiple alanine substitutions. Using similar methods, we also determined the presence of anti-TSP2-8 and CUB1-2 domain antibodies in this cohort of patients. RESULTS: Antibody profiling revealed that anti-ADAMTS13 immunoglobulin G1 and immunoglobulin G4 predominate in plasma of patients with acquired thrombotic thrombocytopenic purpura. Analysis of anti-spacer domain antibodies revealed that Arg568 and Phe592, in addition to residues Arg660, Tyr661, and Tyr665, also contribute to an antigenic surface in the spacer domain. The majority of patients (90%) lost reactivity towards the spacer domain following introduction of multiple alanine substitutions at Arg568, Phe592, Arg660, Tyr661 and Tyr665. Anti-TSP2-8 and anti-CUB1-2 domain-directed antibodies were present in, respectively, 17% and 35% of the patients' samples analyzed. CONCLUSIONS: Immunoglobulin G directed towards a single antigenic surface comprising residues Arg568, Phe592, Arg660, Tyr661 and Tyr665 predominates in the plasma of patients with acquired thrombotic thrombocytopenic purpura.

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F.

Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9-EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9-EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9-EWI-F with potential CD9 homo-dimerization at either end provides a "concatenation model" for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

Amino acid regions 572-579 and 657-666 of the spacer domain of ADAMTS13 provide a common antigenic core required for binding of antibodies in patients with acquired TTP.

Antibodies directed against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). We have previously localized a major antigenic determinant within the spacer domain of ADAMTS13. To identify the amino acid residues of the spacer domain that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of fifteen hybrids (designated A-O) in which 5-10 amino acids of the spacer domain were exchanged for the corresponding region of ADAMTS1. Plasma from six patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of amino acid residues 572-579 (hybrid C) and 657-666 (hybrid M) completely abolished the binding of antibodies from all six patients analyzed. Regions 580-587 (D), 602-620 (G, H), 629-638 (J), and 667-767 (N) contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). Antibodies derived from patient 1 required region 602-620 (G, H) for binding (CGHM-epitope). For antibodies of patient 3, residues 564-571 (B), 580-587 (D), and 629-638 (J) were required (BCDJM-epitope), whereas replacement of residues 602-610 (G) and 629-638 (J) greatly diminished binding of antibodies from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of patients, our results suggest that residues 572-579 (C) and 657-666 (M) comprise a common antigenic core region that is crucial for binding of anti-ADAMTS13 antibodies. Other regions that spatially surround this antigenic core further modulate binding of antibodies to the spacer domain.

Binding mode prediction of cytochrome p450 and thymidine kinase protein-ligand complexes by consideration of water and rescoring in automated docking.

The popular docking programs AutoDock, FlexX, and GOLD were used to predict binding modes of ligands in crystallographic complexes including X-ray water molecules or computationally predicted water molecules. Isoenzymes of two different enzyme systems were used, namely cytochromes P450 (n = 19) and thymidine kinases (n = 19) and three different "water" scenarios: i.e., docking (i) into water-free active sites, (ii) into active sites containing crystallographic water molecules, and (iii) into active sites containing water molecules predicted by a novel approach based on the program GRID. Docking accuracies were determined in terms of the root-mean-square deviation (RMSD) accuracy and, newly defined, in terms of the ligand catalytic site prediction (CSP) accuracy. Consideration of both X-ray and predicted water molecules and the subsequent pooling and rescoring of all solutions (generated by all three docking programs) with the SCORE scoring function significantly improved the quality of prediction of the binding modes both in terms of RMSD and CSP accuracy.

Molecular mechanisms for contribution of MHC molecules to autoimmune diseases.

It will soon be 50 years since the first MHC associations with human disease were described. These seminal studies opened a flourishing area of research, yet much remains to be discovered. Genome-wide association studies of autoimmune diseases have demonstrated that the MHC region has effect sizes that supersede those for any non-MHC locus for most diseases. Thus, an understanding of how particular MHC alleles confer susceptibility will be essential for a comprehensive understanding of autoimmune disease pathogenesis. Here we review recent exciting findings in this important field.