T-cell based immunity is mediated through specific T cell receptor (TCR) recognition of a small antigenic peptide in complex with a host immune molecule, major histocompatibility complex (pMHC). The ...interaction of a TCR and its pMHC ligand is generally quite weak, degenerate and biophysically unfavorable. Yet, the resulting immune response is extremely effective, being both sensitive and specific.
Recent observations indicate that the TCR is an anisotropic mechanosensor. The force sensed by TCR’s recognition module is transmitted to the non-covalently associated signal transduction module. Multiple biophysical methods reveal that the molecular mechanism for TCR-pMHC interaction under force required to induce T cell signaling is linked to “catch bond” formation between a TCR and its cognate ligand pMHC. This kind of dynamic non-covalent bond actually increases the bond lifetime by deforming the molecule to make the interaction lock tighter. The key observation is that the more stimulatory the antigenic peptide, the more pronounced the catch bond and immune response. By contrast, an unrelated, non-antigenic peptide presented by the same MHC molecule does not form a catch bond, instead manifesting a slip bond associated with rapid TCR-pMHC dissociation. In summary, a weak interaction between a TCR and agonist ligand will be dramatically amplified by a catch bond under physical load generated by cell movement during immune surveillance.
These new biophysical concepts, TCR mechanosensor and dynamic catch bond formation, begin to reveal how bioforces tune T cell signaling and should be potentially enlightening for immunotherapy design against cancers.
Summary
Self versus non‐self discrimination is at the core of T‐lymphocyte recognition. To this end, αβ T‐cell receptors (TCRs) ligate ‘foreign’ peptides bound to major histocompatibility complex ...(MHC) class I or class II molecules (pMHC) arrayed on the surface of antigen‐presenting cells (APCs). Since the discovery of TCRs approximately 30 years ago, considerable structural and functional data have detailed the molecular basis of their extraordinary ligand specificity and sensitivity in mediating adaptive T‐cell immunity. This review focuses on the structural biology of the Fab‐like TCRαβ clonotypic heterodimer and its unique features in conjunction with those of the associated CD3εγ and CD3εδ heterodimeric molecules, which, along with CD3ζζ homodimer, comprise the TCR complex in a stoichiometry of 1:1:1:1. The basis of optimized TCRαβ docking geometry on the pMHC linked to TCR mechanotransduction and required for T‐cell signaling as well as CD4 and CD8 co‐receptor function is detailed. A model of the TCR ectodomain complex including its connecting peptides suggests how force generated during T‐cell immune surveillance and at the immunological synapse results in dynamic TCR quaternary change involving its heterodimeric components. Potential insights from the structural biology relevant to immunity and immunosuppression are revealed.
Significance The αβ T-cell receptor (TCR) on mammalian T lymphocytes recognizes intracellular pathogens to afford protective immunity. Detection of various foreign peptides bound to MHC molecules as ...TCR ligands occurs during immune surveillance where mechanical forces are generated through cell movement. Using single-molecule optical tweezer assays, we show with isolated and complete receptors on single T cells that both sensitivity and specificity of the biological T-lymphocyte response is dependent upon force-based interactions. Our work demonstrates a catch-and-release αβTCR structural conversion correlating with ligand potency wherein a strongly binding/compact state transitions to a weakly binding/extended state. An allosteric mechanism controls bond strength and lifetime, supporting a model in which quaternary αβTCR subunit associations regulate TCR recognition under load.
The αβ T-cell receptor (TCR) on each T lymphocyte mediates exquisite specificity for a particular foreign peptide bound to a major histocompatibility complex molecule (pMHC) displayed on the surface of altered cells. This recognition stimulates protection in the mammalian host against intracellular pathogens, including viruses, and involves piconewton forces that accompany pMHC ligation. Physical forces are generated by T-lymphocyte movement during immune surveillance as well as by cytoskeletal rearrangements at the immunological synapse following cessation of cell migration. The mechanistic explanation for how TCRs distinguish between foreign and self-peptides bound to a given MHC molecule is unclear: peptide residues themselves comprise few of the TCR contacts on the pMHC, and pathogen-derived peptides are scant among myriad self-peptides bound to the same MHC class arrayed on infected cells. Using optical tweezers and DNA tether spacer technology that permit piconewton force application and nanometer scale precision, we have determined how bioforces relate to self versus nonself discrimination. Single-molecule analyses involving isolated αβ-heterodimers as well as complete TCR complexes on T lymphocytes reveal that the FG loop in the β-subunit constant domain allosterically controls both the variable domain module’s catch bond lifetime and peptide discrimination via force-driven conformational transition. In contrast to integrins, the TCR interrogates its ligand via a strong force-loaded state with release through a weakened, extended state. Our work defines a key element of TCR mechanotransduction, explaining why the FG loop structure evolved for adaptive immunity in αβ but not γδTCRs or immunoglobulins.
Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors, and co-factors for signaling. ...Recently, genetic studies suggested that netrin is involved in neuronal haptotaxis, which requires a reversible adhesion process. Structural data indicate that netrin can also mediate trans-adhesion between apposing cells decorated with its receptors on the condition that the auxiliary guidance cue draxin is present. Here, we propose that netrin is involved in conditional adhesion, a reversible and localized process that can contribute to cell adhesion and migration. We suggest that netrin-mediated adhesion and signaling are linked, and that local environmental factors in the ventricular zone, the floor plate, or other tissues coordinate its function.
Structural studies reveal molecular details of netrin-mediated clustering of DCC and uncoordinated 5 (UNC5), suggesting how they switch between growth cone attraction and repulsion in axon guidance.Three separate DCC-binding sites have been identified on netrin, and each site can be targeted by competing receptors or cofactors.Environmental cofactors may affect netrin-mediated signaling and adhesion.Structural studies suggest that the auxiliary guidance cue draxin locally expressed in the spinal cord alters netrin–DCC interactions to promote adhesion and axonal fasciculation.We propose that netrin mediates conditional adhesion between cells decorated with netrin receptors. Conditional adhesion is a reversible and highly localized process that can facilitate fasciculation, haptotaxis, and downstream signaling.
Recognition by the leukocyte integrins αXβ2 and αMβ2 of complement iC3b-opsonized targets is essential for effector functions including phagocytosis. The integrin-binding sites on iC3b remain ...incompletely characterized. Here, we describe negative-stain electron microscopy and biochemical studies of αXβ2 and αMβ2 in complex with iC3b. Despite high homology, the two integrins bind iC3b at multiple distinct sites. αXβ2 uses the αX αI domain to bind iC3b on its C3c moiety at one of two sites: a major site at the interface between macroglobulin (MG) 3 and MG4 domains, and a less frequently used site near the C345C domain. In contrast, αMβ2 uses its αI domain to bind iC3b at the thioester domain and simultaneously interacts through a region near the αM β-propeller and β2 βI domain with a region of the C3c moiety near the C345C domain. Remarkably, there is no overlap between the primary binding site of αXβ2 and the binding site of αMβ2 on iC3b. Distinctive binding sites on iC3b by integrins αXβ2 and αMβ2 may be biologically beneficial for leukocytes to more efficiently capture opsonized pathogens and to avoid subversion by pathogen factors.
The inside-out signaling of inte- grins regulates the ligand-binding affinity of the cell surface receptors in response to changes in the environ- ment for cell survival. The specific binding to the ...cytoplasmic tail of in- tegrin＇s β subunit by the intracellular protein talin is the key step of inside- out signaling. A ＂pull-push＂ mecha- nism has been proposed to explain how the PIP2-enriched membrane disrupts the dual auto-inhibition of the N-terminal talin-FERM domain by the C-terminal talin-rod domain such that activated talin-FERM can reach the β-tail for integrin activa- tion.
Drugs that can protect against organ damage are urgently needed, especially for diseases such as sepsis and brain stroke. We discovered that terazosin (TZ), a widely marketed α1-adrenergic receptor ...antagonist, alleviated organ damage and improved survival in rodent models of stroke and sepsis. Through combined studies of enzymology and X-ray crystallography, we discovered that TZ binds a new target, phosphoglycerate kinase 1 (Pgk1), and activates its enzymatic activity, probably through 2,4-diamino-6,7-dimethoxyisoquinoline's ability to promote ATP release from Pgk1. Mechanistically, the ATP generated from Pgk1 may enhance the chaperone activity of Hsp90, an ATPase known to associate with Pgk1. Upon activation, Hsp90 promotes multistress resistance. Our studies demonstrate that TZ has a new protein target, Pgk1, and reveal its corresponding biological effect. As a clinical drug, TZ may be quickly translated into treatments for diseases including stroke and sepsis.
Vaccines targeting HIV-1's gp160 spike protein are stymied by high viral mutation rates and structural chicanery. gp160's membrane-proximal external region (MPER) is the target of naturally arising ...broadly neutralizing antibodies (bnAbs), yet MPER-based vaccines fail to generate bnAbs. Here, nanodisc-embedded spike protein was investigated by cryo-electron microscopy and molecular-dynamics simulations, revealing spontaneous ectodomain tilting that creates vulnerability for HIV-1. While each MPER protomer radiates centrally towards the three-fold axis contributing to a membrane-associated tripod structure that is occluded in the upright spike, tilting provides access to the opposing MPER. Structures of spike proteins with bound 4E10 bnAb Fabs reveal that the antibody binds exposed MPER, thereby altering MPER dynamics, modifying average ectodomain tilt, and imposing strain on the viral membrane and the spike's transmembrane segments, resulting in the abrogation of membrane fusion and informing future vaccine development.
Abstract
The nucleoprotein (NP) of influenza virus is the core component of the ribonucleoprotein (RNP) and performs multiple structural and functional roles. Structures of the influenza A, B and D ...NP molecules have been solved previously, but structural information on how NP interacts with RNA remains elusive. Here we present the crystal structure of an obligate monomer of H5N1 NP in complex with RNA nucleotides to 2.3 Å, and a C-terminal truncation of this mutant, also in complex with RNA nucleotides, to 3 Å. In both structures, three nucleotides were identified near two positive grooves of NP suggested to be important for RNA binding. Structural evidence supports that conformational changes of flexible loops and the C-terminal tail both play important roles in the binding of RNA. Based on the structure, we propose a mechanism by which NP captures RNA by flexible loops and transfers it onto the positive binding grooves. Binding of RNA by NP is a crucial step for template re-encapsidation during transcription and replication and cRNP formation. Our structures thus provide insights into the molecular virology of the influenza virus.
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