Massimo Degano

An αβ T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex

The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides bound to major histocompatibility complex (MHC) molecules by the αβ T cell receptor (TCR). The x-ray structure of the complete extracellular fragment of a glycosylated αβ TCR was determined at 2.5 angstroms, and its orientation bound to a class I MHC-peptide (pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable Vα and Vβ domains but deviates in the constant Cα domain and in the interdomain pairing of Cα with Cβ. Four of seven possible asparagine-linked glycosylation sites have ordered carbohydrate moieties, one of which lies in the Cα-Cβ interface. The TCR combining site is relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3s (complementarity-determining regions) of the α and β chains. The 2C TCR covers the class I MHC H-2Kb binding groove so that the Vα CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the Vβ chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the Vα and Vβ CDR3s straddle the peptide between the helices around the central position of the peptide.

Structural Basis of 2C TCR Allorecognition of H-2Ld Peptide Complexes

MHC class I H-2Ld complexed with peptide QL9 (or p2Ca) is a high-affinity alloantigen for the 2C TCR. We used the crystal structure of H-2Ld with a mixture of bound peptides at 3.1 A to construct a model of the allogeneic 2C-Ld/QL9 complex for comparison with the syngeneic 2C-Kb/dEV8 structure. A prominent ridge on the floor of the Ld peptide-binding groove, not present in Kb, creates a C-terminal bulge in Ld peptides that greatly increases interactions with the 2C beta-chain. Furthermore, weak electrostatic complementarity between Asp77 on the alpha1 helix of Kb and 2C is enhanced in the allogeneic complex by closer proximity of QL9 peptide residue AspP8 to the 2C HV4 loop.

Crystal Structure of Mouse CD1d Bound to the Self Ligand Phosphatidylcholine: A Molecular Basis for NKT Cell Activation

NKT cells are immunoregulatory lymphocytes whose activation is triggered by the recognition of lipid Ags in the context of the CD1d molecules by the TCR. In this study we present the crystal structure to 2.8 Å of mouse CD1d bound to phosphatidylcholine. The interactions between the ligand acyl chains and the CD1d molecule define the structural and chemical requirements for the binding of lipid Ags to CD1d. The orientation of the polar headgroup toward the C terminus of the α1 helix provides a rationale for the structural basis for the observed Vα chain bias in invariant NKT cells. The contribution of the ligand to the protein surface suggests a likely mode of recognition of lipid Ags by the NKT cell TCR.

Crystal structures of soybean beta-amylase reacted with beta-maltose and maltal: active site components and their apparent roles in catalysis.

The crystal structures of catalytically competent soybean beta-amylase, unliganded and bathed with small substrates (beta-maltose, maltal), were determined at 1.9-2.2-A resolution. Two molecules of beta-maltose substrate bind to the protein in tandem, with some maltotetraose enzymic condensation product sharing the same binding sites. The beta-amylase soaked with maltal shows a similar arrangement of two bound molecules of 2-deoxymaltose, the enzymic hydration product. In each case the nonreducing ends of the saccharide ligands are oriented toward the base of the proteins active site pocket. The catalytic center, located between the bound disaccharides and found deeper in the pocket than where the inhibitor alpha-cyclodextrin binds, is characterized by the presence of oppositely disposed carboxyl groups of two conserved glutamic acid residues. The OE2 carboxyl of Glu 186 is below the plane of the penultimate glucose residue (Glc 2) of bound maltotetraose, 2.6 A from the oxygen atom of that ligands penultimate alpha-1,4-glucosidic linkage. The OE2 carboxyl of Glu 380 lies above the plane of Glc 2, 2.8 A from the O-1 atom of the more deeply bound beta-maltose. Saccharide binding does not alter the spatial coordinates of these two carboxyl groups or the overall conformation of the 57-kDa protein. However, the saccharide complexes of the active enzyme are associated with a significant (10 A) local conformational change in a peptide segment of a loop (L3) that borders the active site pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure of human ERp44 shows a dynamic functional modulation by its carboxy-terminal tail

ERp44 mediates thiol‐dependent retention in the early secretory pathway, forming mixed disulphides with substrate proteins through its conserved CRFS motif. Here, we present its crystal structure at a resolution of 2.6 Å. Three thioredoxin domains—a, b and b′—are arranged in a clover‐like structure. A flexible carboxy‐terminal tail turns back to the b′ and a domains, shielding a hydrophobic pocket in domain b′ and a hydrophobic patch around the CRFS motif in domain a. Mutational and functional studies indicate that the C‐terminal tail gates the CRFS area and the adjacent hydrophobic pocket, dynamically regulating protein quality control.

Fine specificity of TCR complementarity-determining region residues and lipid antigen hydrophilic moieties in the recognition of a CD1-lipid complex.

αβ TCR can recognize peptides presented by MHC molecules or lipids and glycolipids presented by CD1 proteins. Whereas the structural basis for peptide/MHC recognition is now clearly understood, it is not known how the TCR can interact with such disparate molecules as lipids. Recently, we demonstrated that the αβ TCR confers specificity for both the lipid Ag and CD1 isoform restriction, indicating that the TCR is likely to recognize a lipid/CD1 complex. We hypothesized that lipids may bind to CD1 via their hydrophobic alkyl and acyl chains, exposing the hydrophilic sugar, phosphate, and other polar functions for interaction with the TCR complementarity-determining regions (CDRs). To test this model, we mutated the residues in the CDR3 region of the DN1 TCR β-chain that were predicted to project between the CD1b α helixes in a model of the TCR/CD1 complex. In addition, we tested the requirement for the negatively charged and polar functions of mycolic acid for Ag recognition. Our findings indicate that the CDR loops of the TCR form the Ag recognition domain of CD1-restricted TCRs and suggest that the hydrophilic domains of a lipid Ag can form a combinatorial epitope recognized by the TCR.

A pH-Regulated Quality Control Cycle for Surveillance of Secretory Protein Assembly

Summary To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44’s active cysteine, simultaneously unmask the substrate binding site and −RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles.

Dalton communications. Synthesis and structure of the first phosphine oxide complex of copper(I): evidence for a marked ‘borderline’ character of the metal centre

The remarkable stability of [Cu(dppf)(odppf)]BF4[dppf = 1,1′-bis(diphenylphosphino)ferrocene, odppf = 1,1′-bis(oxodiphenylphosphoranyl)ferrocene], characterized in the solid state by X-ray analysis and in solution by 31P NMR spectroscopy, reveals a marked borderline character of copper(I).

IGHV gene insertions and deletions in chronic lymphocytic leukemia: "CLL-biased" deletions in a subset of cases with stereotyped receptors.

Nucleotide insertions/duplications or deletions in immunoglobulin heavy chain genes have been found in 24/760 patients (3.15%) with chronic lymphocytic leukemia (CLL). In 21/24 cases, the inserted/duplicated or lost nucleotides occurred in multiples of 3; therefore, the original reading frame was maintained and a potentially intact receptor was coded. The pattern and location of insertions/duplications or deletions in CLL and their restriction to mutated IGHV rearranged genes strongly suggests that they resulted from somatic hypermutation. Their incidence in CLL is consistent with previous reports in normal, auto‐reactive and neoplastic human B cells, thus seemingly indicating that these modifications generally arise without any particular disease‐specific associations. A striking exception to this rule was identified in CLL IGHV3‐21‐expressing cases: one amino acid was deleted from the CDR2 region in 16/63 (25.4%) mutated CLL IGHV3‐21 sequences (including public database‐derived IGHV3‐21 CLL cases + the present series) vs. only 2/257 (0.78%) public database‐derived mutated non‐CLL IGHV3‐21 sequences; 15/16 CLL IGHV3‐21 sequences carrying this deletion belonged to a subset with unique, shared HCDR3 and light chain CDR3 motifs. This finding further supports the idea of selective antigenic pressures playing a pathogenetic role in some CLL cases.

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells

Summary: Activation of T cells involves multiple receptor‐ligand interactions between T cells and antigen presenting cells (APC), At least two signals are required for T‐cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T‐cell receptors (TCR). whereas Signal 2 is induced by the interactions of co‐stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal I to T cells remains unclear. The effects of Signal 1 and Signal 2 on T‐cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal I alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)‐l can provide the second signal for CD8 T‐cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and [CAM‐1 on the same APC. Thus, the data suggest that at least two qualitatively different co‐stimulation signals are required for full activation of CD8 T cells under physiological conditions.