Regulation of membrane receptor mobility tunes cellular response to external signals, such as in binding of B cell receptors (BCR) to antigen, which initiates signaling. However, whether BCR signaling is regulated by BCR mobility, and what factors mediate this regulation, are not well understood. Here we use single molecule imaging to examine BCR movement during signaling activation and a novel machine learning method to classify BCR trajectories into distinct diffusive states. Inhibition of actin dynamics downstream of the actin nucleating factors, Arp2/3 and formin, decreases BCR mobility. Constitutive loss or acute inhibition of the Arp2/3 regulator, N-WASP, which is associated with enhanced signaling, increases the proportion of BCR trajectories with lower diffusivity. Furthermore, loss of N-WASP reduces the diffusivity of CD19, a stimulatory co-receptor, but not that of Fc
T cell and B cell antigen receptors share a conserved core transmembrane structure
The B cell and T cell antigen receptors (BCR and TCR) share a common architecture in which variable dimeric antigen-binding modules assemble with invariant dimeric signaling modules to form functional receptor complexes. In the TCR, a highly conserved T cell receptor αβ (TCRαβ) transmembrane (TM) interface forms a rigid structure around which its three dimeric signaling modules assemble through well-characterized polar interactions. Noting that the key features stabilizing this TCRαβ TM interface also appear with high evolutionary conservation in the TM sequences of the membrane immunoglobulin (mIg) heavy chains that form the BCR’s homodimeric antigen-binding module, we asked whether the BCR contained an analogous TM structure. Using an unbiased biochemical and computational modeling approach, we found that the mouse IgM BCR forms a core TM structure that is remarkably similar to that of the TCR. This structure is reinforced by a network of interhelical hydrogen bonds, and our model is nearly identical to the arrangement observed in the just-released cryo-electron microscopy (cryo-EM) structures of intact human BCRs. Our biochemical analysis shows that the integrity of this TM structure is vital for stable assembly with the BCR signaling module CD79AB in the B cell endoplasmic reticulum, and molecular dynamics simulations indicate that BCRs of all five isotypes can form comparable structures. These results demonstrate that, despite their many differences in composition, complexity, and ligand type, TCRs and BCRs rely on a common core TM structure that has been shaped by evolution for optimal receptor assembly and stability in the cell membrane.
more »
« less
- Award ID(s):
- 2111728
- NSF-PAR ID:
- 10426966
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 119
- Issue:
- 48
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract γ RIIB, an inhibitory co-receptor. Our results implicate a dynamic actin network in fine-tuning receptor mobility and receptor-ligand interactions for modulating B cell signaling. -
Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates.more » « less
-
Lavrik, Inna (Ed.)more » « less
T cells form transient cell-to-cell contacts with antigen presenting cells (APCs) to facilitate surface interrogation by membrane bound T cell receptors (TCRs). Upon recognition of molecular signatures (antigen) of pathogen, T cells may initiate an adaptive immune response. The duration of the T cell/APC contact is observed to vary widely, yet it is unclear what constructive role, if any, such variations might play in immune signaling. Modeling efforts describing antigen discrimination often focus on steady-state approximations and do not account for the transient nature of cellular contacts. Within the framework of a kinetic proofreading (KP) mechanism, we develop a stochastic
First Receptor Activation Model (FRAM) describing the likelihood that a productive immune signal is produced before the expiry of the contact. Through the use of extreme statistics, we characterize the probability that the first TCR triggering is induced by a rare agonist antigen and not by that of an abundant self-antigen. We show that defining positive immune outcomes as resilience to extreme statistics and sensitivity to rare events mitigates classic tradeoffs associated with KP. By choosing a sufficient number of KP steps, our model is able to yield single agonist sensitivity whilst remaining non-reactive to large populations of self antigen, even when self and agonist antigen are similar in dissociation rate to the TCR but differ largely in expression. Additionally, our model achieves high levels of accuracy even when agonist positive APCs encounters are rare. Finally, we discuss potential biological costs associated with high classification accuracy, particularly in challenging T cell environments. -
ABSTRACT Tsr, the serine chemoreceptor in Escherichia coli , transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associate in vivo with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture. IMPORTANCE In Escherichia coli , membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of the E. coli serine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.more » « less
-
Protein–protein binding domains are critical in signaling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain–containing proteins has tandem domains, which are thought to enhance binding affinity and specificity. However, a trade-off exists between long-lived binding and the ability to rapidly reverse signaling, which is a critical requirement of noise-filtering mechanisms such as kinetic proofreading. Here, we use modeling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases. Using surface plasmon resonance, we show that the phosphatase CD45 can accelerate the unbinding rate of zeta chain–associated protein kinase 70 (ZAP70), a tandem SH2 domain–containing kinase, from biphosphorylated peptides from the T cell receptor (TCR). An important functional prediction of accelerated unbinding is that the intracellular ZAP70–TCR half-life in T cells will not be fixed but rather, dependent on the extracellular TCR–antigen half-life, and we show that this is the case in both cell lines and primary T cells. The work highlights that tandem SH2 domains can break the trade-off between signal fidelity (requiring long half-life) and signal reversibility (requiring short half-life), which is a key requirement for T cell antigen discrimination mediated by kinetic proofreading.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2208058119
