Matthew E. Call

The Organizing Principle in the Formation of the T Cell Receptor-CD3 Complex

The T cell receptor (TCR) serves a critical function in the immune system and represents one of the most complex receptor structures. A striking feature is the presence of nine highly conserved, potentially charged residues in the transmembrane helices. Previous models have attempted to explain assembly based on pairwise interactions of these residues. Using a novel method for the isolation of intact radiolabeled protein complexes, we demonstrate that one basic and two acidic transmembrane residues are required for the assembly of each of the three signaling dimers with the TCR. This remarkable three-helix arrangement applies to all three assembly steps and represents the organizing principle for the formation of this intricate receptor structure.

Regulation of T Cell Receptor Activation by Dynamic Membrane Binding of the CD3ɛ Cytoplasmic Tyrosine-Based Motif

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes

PA28 is a γ‐interferon‐induced complex that associates with the 20S proteasome and stimulates breakdown of small peptides. Recent immunoprecipitation studies indicate that, in vivo, PA28 also exists in larger complexes that also contain the 19S particle, which is required for ATP‐ubiquitin‐dependent degradation of proteins. However, because of its lability, the structure and properties of this larger complex remain unclear. Here, we demonstrate that, in vitro, PA28 can associate with singly capped’ 26S (i.e. 19S–20S) proteasomes. Electron microscopy of the resulting structures revealed one PA28 ring at one end of the 20S particle and a 19S complex at the other. These hybrid complexes show enhanced hydrolysis of small peptides, but no significant increase in rates of protein breakdown. Nevertheless, during breakdown of proteins, the complexes containing PA28αβ or PA28α generated a pattern of peptides different from those generated by 26S proteasomes, without altering mean product length. Presumably, this change in peptides produced accounts for the capacity of PA28 to enhance antigen presentation.

The Structure of the ζζ Transmembrane Dimer Reveals Features Essential for Its Assembly with the T Cell Receptor

The T cell receptor (TCR) alphabeta heterodimer communicates ligand binding to the cell interior via noncovalently associated CD3gammaepsilon, CD3deltaepsilon, and zetazeta dimers. While structures of extracellular components of the TCR-CD3 complex are known, the transmembrane (TM) domains that mediate assembly have eluded structural characterization. Incorporation of the zetazeta signaling module is known to require one basic TCRalpha and two zetazeta aspartic acid TM residues. We report the NMR structure of the zetazeta(TM) dimer, a left-handed coiled coil with substantial polar contacts. Mutagenesis experiments demonstrate that three polar positions are critical for zetazeta dimerization and assembly with TCR. The two aspartic acids create a single structural unit at the zetazeta interface stabilized by extensive hydrogen bonding, and there is evidence for a structural water molecule (or molecules) within close proximity. This structural unit, representing only the second transmembrane dimer interface solved to date, serves as a paradigm for the assembly of all modules involved in TCR signaling.

Antibodies from Inflamed Central Nervous System Tissue Recognize Myelin Oligodendrocyte Glycoprotein

Autoantibodies to myelin oligodendrocyte glycoprotein (MOG) can induce demyelination and oligodendrocyte loss in models of multiple sclerosis (MS). Whether anti-MOG Abs play a similar role in patients with MS or inflammatory CNS diseases by epitope spreading is unclear. We have therefore examined whether autoantibodies that bind properly folded MOG protein are present in the CNS parenchyma of MS patients. IgG was purified from CNS tissue of 14 postmortem cases of MS and 8 control cases, including cases of encephalitis. Binding was assessed using two independent assays, a fluorescence-based solid-phase assay and a solution-phase RIA. MOG autoantibodies were identified in IgG purified from CNS tissue by solid-phase immunoassay in 7 of 14 cases with MS and 1 case of subacute sclerosing panencephalitis, but not in IgG from noninflamed control tissue. This finding was confirmed with a solution-phase RIA, which measures higher affinity autoantibodies. These data demonstrate that autoantibodies recognizing MOG are present in substantially higher concentrations in the CNS parenchyma compared with cerebrospinal fluid and serum in subjects with MS, indicating that local production/accumulation is an important aspect of autoantibody-mediated pathology in demyelinating CNS diseases. Moreover, chronic inflammatory CNS disease may induce autoantibodies by virtue of epitope spreading.

The structural basis for intramembrane assembly of an activating immunoreceptor complex

Many receptors that activate cells of the immune system are multisubunit membrane protein complexes in which ligand recognition and signaling functions are contributed by separate protein modules. Receptors and signaling subunits assemble through contacts among basic and acidic residues in their transmembrane domains to form the functional complexes. Here we report the nuclear magnetic resonance (NMR) structure of the membrane-embedded, heterotrimeric assembly formed by association of the DAP12 signaling module with the natural killer (NK) cell–activating receptor NKG2C. The main intramembrane contact site is formed by a complex electrostatic network involving five hydrophilic transmembrane residues. Functional mutagenesis demonstrated that similar polar intramembrane motifs are also important for assembly of the NK cell–activating NKG2D-DAP10 complex and the T cell antigen receptor (TCR)–invariant signaling protein CD3 complex. This structural motif therefore lies at the core of the molecular organization of many activating immunoreceptors.

Stoichiometry of the T-cell receptor-CD3 complex and key intermediates assembled in the endoplasmic reticulum

The T‐cell receptor (TCR)–CD3 complex is critical for T‐cell development and function, and represents one of the most complex transmembrane receptors. Models of different stoichiometry and valency have been proposed based on cellular experiments and these have important implications for the mechanisms of receptor triggering. Since determination of receptor stoichiometry in T‐cells is not possible due to the presence of previously synthesized, unlabeled receptor components with different half‐lives, we examined the stoichiometry of the receptor assembled in endoplasmic reticulum (ER) microsomes of B‐cell origin. The stoichiometric relationship among all subunits was directly determined using intact radiolabeled TCR–CD3 complexes that were isolated with a sequential, non‐denaturing immunoprecipitation method, and identical results were obtained with two detergents belonging to different structural classes. The results firmly establish that the αβ TCR–CD3 complex assembled in the ER is monovalent and composed of one copy of the TCRαβ, CD3δε, CD3γε and ζ−ζ dimers.

Common themes in the assembly and architecture of activating immune receptors

Each of the many different cell types of the immune system expresses one or several activating receptors which serve a central role in the cells surveillance function. Many of these cell-surface receptors share a distinctive modular design that consists of a ligand-binding module with no intrinsic signalling capability that is non-covalently associated with one or more dimeric signalling modules. Receptor assembly is directed by unique polar contacts within the transmembrane domains, whereas extracellular contacts can contribute to stability and specificity. This Review discusses the structural basis of receptor assembly and the implications of these findings for the mechanisms of receptor triggering.

Dominant-negative effect of the heterozygous C104R TACI mutation in common variable immunodeficiency (CVID)

B cells from patients with common variable immunodeficiency (CVID) who are heterozygous for transmembrane activator and CAML interactor (TACI) mutation C104R, which abolishes ligand binding, fail to produce Igs in response to TACI ligand. It is not known whether this is due to haploinsufficiency or dominant interference. Using in vitro transfection assays, here we demonstrate that C104R and the corresponding murine TACI mutant, C76R, which also does not bind ligand, dominantly interfere with TACI signaling. This effect was dependent on preassociation of the mutants with WT TACI in the absence of ligand. The mutants did not interfere with ligand binding by WT TACI, suggesting that they may act by disrupting ligand-induced receptor rearrangement and signaling. This work demonstrates that TACI preassembles as an oligomeric complex prior to ligand binding and provides a mechanistic insight into how the heterozygous C104R TACI mutation can potentially lead to CVID.

Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling

The T-cell receptor (TCR)-CD3 complex serves as a central paradigm for general principles of receptor assembly, ligand recognition, and signaling in the immune system. There is no other receptor system that matches the diversity of both receptor and ligand components. The recent expansion of the immunological structural database is beginning to identify key principles of MHC and peptide recognition. The multicomponent assembly of the TCR complex illustrates general principles used by many receptors in the immune system, which rely on basic and acidic transmembrane residues to guide assembly. The intrinsic binding of the cytoplasmic domains of the CD3epsilon and zeta chains to the inner leaflet of the plasma membrane represents a novel mechanism for control of receptor activation: Insertion of critical CD3epsilon tyrosines into the hydrophobic membrane core prevents their phosphorylation before receptor engagement.