Summary Antigen recognition of peptide-major histocompatibility complexes (pMHCs) by T-cells, a key step in initiating adaptive immune responses, is performed by the T-cell receptor (TCR) bound to CD3 heterodimers. However, the biophysical basis of the transmission of TCR-CD3 extracellular interaction into a productive intracellular signaling sequence remains incomplete. Herein, we used nuclear magnetic resonance (NMR) spectroscopy combined with mutational analysis and computational docking to derive a structural model of the extracellular TCR-CD3 assembly. In the inactivated state, CD3γε interacts with the helix-3 and helix 4-F strand regions of the TCR Cβ subunit while CD3δε interacts with the F and C strand regions of TCR Cα subunit in this model, thereby placing the CD3 subunits on opposing sides of the TCR. Together this work identifies the molecular contacts between the TCR and CD3 subunits thereby identifying a physical basis for transmitting an activating signal through the complex.

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-B and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15 N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding ␣2-␣3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the ␣2-␣3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the ␣2-␣3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.Inflammatory caspases such as caspase-1 play an essential role in innate immune responses to infection by regulating the processing of pro-inflammatory cytokines interleukin-1␤ and interleukin-18 into their mature, secreted forms (1, 2). Tight regulation of the production of these cytokines is required to maintain the homeostasis of host tissues. Excessive interleukin-1␤ production and chronic inflammation are hallmarks of many autoimmune diseases that present both systemically and within the central nervous system, including rheumatoid arthritis and multiple sclerosis (3, 4).Similar to initiator caspases involved in apoptosis, the activation of inflammatory caspases requires their recruitment into a multiprotein signaling complex, which promotes dimerization and cleavage to produce the active enzyme via an induced proximity mechanism (2, 5). Recently, models for caspase-1 activation have been proposed whereby inflammatory stimuli promote the formation of molecular platforms referred to as inflammasomes (6). The NALP1 inflammasome induces the activation of both caspase-1 and caspase-5 through the formation of a complex that also contains the proteins NALP1 and ASC (7). Similarly, the NALP2/NALP3 inflammasome is involved in the activation of caspase-1 through the r...

The T-cell receptor (TCR) complex comprises TCRαβ, CD3γε, CD3δε, and CD3ζζ. TCRαβ engagement with peptide-bound major histocompatibility complex (pMHC) triggers CD3 phosphorylation, which is regulated by mechanical force. However, the inner workings of the TCR mechanotransduction machinery remains unclear. TCR ectodomain (ECD) interactions have been inferred from structural and mutagenesis studies. Due to their extreme weakness, however, direct measurements of affinity had failed and of force regulation have never been attempted. Here we measured two-dimensional affinities and force-dependent lifetimes of interactions among TCRαβ, CD3γε, and CD3δε ECDs, showing a cooperative CD3δε–TCRαβ–CD3γε catch bond with longer-lasting lifetime that exceeds the TCR–pMHC bond lifetime. Molecular dynamics stimulations revealed a central interacting region surrounded by TCR ECDs and identified critical interacting residues at their interfaces. Interfering TCR ECD interactions by antibodies impaired TCR–pMHC interaction and T cell function. Mutating residues that mediate TCR ECD cis-interactions with CD3s altered the catch bond of TCR–pMHC trans-interaction, which correlates with changed T cell cytokine production. Thus, TCR mechanotransduction is supported by cooperative TCR ECD interactions, which regulates T cell function. Our results provide a missing link between pMHC ligation and CD3 signaling and may guide future TCR engineering design for immunotherapies.

The TCR integrates forces in its triggering process upon interaction with pMHC. Force elicits TCR catch-slip bonds with strong pMHCs but slip-only bonds with weak pMHCs. We develop two models and apply them to analyze 55 datasets, demonstrating the models’ ability to quantitatively integrate and classify a broad range of bond behaviors and biological activities. Comparing to a generic two-state model, our models can distinguish class I from class II MHCs and correlate their structural parameters with the TCR/pMHC’s potency to trigger T cell activation. The models are tested by mutagenesis using an MHC and a TCR mutated to alter conformation changes. The extensive comparisons between theory and experiment provide model validation and testable hypothesis regarding specific conformational changes that control bond profiles, thereby suggesting structural mechanisms for the inner workings of the TCR mechanosensing machinery and plausible explanations of why and how force may amplify TCR signaling and antigen discrimination.

The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3′-phosphomonoesterase and 3′-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn2+• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3′-deoxynucleotide, 3′-deoxynucleotide 3′-phosphate, or 3′ ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3′-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd2+) and inhibitory (Zn2+) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn2+.