Summary 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.

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 tumors are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumor microenvironment. We devised an in vivo pooled shRNA screen in which shRNAs targeting negative regulators became highly enriched in tumors by releasing a block on T cell proliferation upon tumor antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumor or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family: In tumors, Ppp2r2d knockdown inhibited T cell apoptosis and enhanced T cell proliferation as well as cytokine production. Key regulators of immune function can thus be discovered in relevant tissue microenvironments.

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 IntroductionVon Willebrand factor (VWF) is a key hemostatic glycoprotein involved in the adhesion of platelets to sites of vascular perturbation. 1 During its biosynthesis in endothelial cells, VWF undergoes a number of posttranslational modifications that include the formation of intermolecular disulfide bonds between the carboxylterminal cysteine knot domains and the amino-terminal D3 domains. 2,3 Current findings suggest that the resulting VWF polymers condense into tubular structures in the trans Golgi network and are subsequently packaged into Weibel-Palade bodies, rod-shaped subcellular organelles. 4,5 Upon release of Weibel-Palade body contents, the VWF tubules rapidly unfold, and the fluid shear stress in the flowing blood induces the formation of ultra-large VWF (UL-VWF) strings on the surface of endothelial cells. 6,7 Andre et al 6 have shown that the appearance of platelet-decorated strings is a transient process in vivo. This transient nature is attributed to the rapid proteolysis of UL-VWF multimers by the metalloprotease ADAMTS13. 8,9 In the absence of ADAMTS13, the rate at which platelet strings disappear from the endothelium is markedly reduced.Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterized by hemolytic anemia, severe thrombocytopenia, and the presence of schistocytes in blood smears and is accompanied by a deficiency in ADAMTS13. ADAMTS13 is a large multidomain protein that consists of a propeptide, a catalytic metalloprotease domain, a disintegrin-like domain, a thrombospondin type I repeat (TSP), a cysteine-rich domain, a spacer domain, 7 additional TSP repeats, and 2 carboxyl-terminal CUB domains. [10][11][12] In the majority of patients with TTP, inhibitory antibodies targeting ADAMTS13 have been found. [13][14][15][16] In addition to inhibition of ADAMTS13 activity, anti-ADAMTS13 antibodies may also accelerate ADAMTS13 clearance. 17 Epitope mapping studies have shown that the cysteine-rich/spacer domains contain the major binding site for human anti-ADAMTS13 antibodies. [18][19][20][21] Additional epitopes for human anti-ADAMTS13 antibodies located outside the spacer domain have also been identified. 18,22,23 In a previous study, we have shown that amino acid residues Y658-Y665 within the spacer domain comprise part of a core binding site for anti-ADAMTS13 antibodies. 24 The ADAMTS13 metalloprotease domain cleaves the VWF A2 domain at the Y1605-M1606 scissile bond. The metalloprotease domain by itself cannot efficiently proteolyse VWF; the proximal ...

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.