The Allostery Model of TCR Regulation Wolfgang W

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The Allostery Model of TCR Regulation Wolfgang W. A. Schamel, Balbino Alarcon, Thomas Höfer and Susana Minguet This information is current as J Immunol 2017; 198:47-52; ; of September 25, 2021. doi: 10.4049/jimmunol.1601661 http://www.jimmunol.org/content/198/1/47 Downloaded from References This article cites 69 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/198/1/47.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Brief Reviews Immunology

The Allostery Model of TCR Regulation x { ‖ Wolfgang W. A. Schamel,*,†,‡ Balbino Alarcon, Thomas Ho¨fer, , and Susana Minguet*,† The activity of the ab TCR is controlled by confor- having come to signify three-dimensionality). Thus, allosteric mational switches. In the resting conformation, the regulation indicates that binding of one ligand at one site of a TCR is not phosphorylated and is inactive. Binding protein, or protein complex, changes the binding of a second of multivalent peptide-MHC to the TCR stabilizes ligand, or a substrate, at a topographically distinct site of the the active conformation, leading to TCR signaling. protein (1, 2). The ﬁrst ligand can enhance or reduce binding of These two conformations allow the TCRs to be allo- the second ligand, being a positive or negative allosteric regu- sterically regulated. We review recent data on hetero- lator, respectively. Hence, allosteric regulation is distinct from competitive inhibition, resulting from substrate and inhibitor tropic allostery where peptide-MHC and membrane Downloaded from cholesterol serve opposing functions as positive and binding to the same site. Switch-like responses typically arise negative allosteric regulators, respectively. In resting from the interaction of multiple subunits in allosteric protein T cells cholesterol keeps TCRs in the resting confor- complexes and thus multiple ligand binding sites. The Monod- Wyman-Changeux(MWC)model,asithascometobecalled, mation that otherwise would become spontaneously has stood the test of time remarkably well, explaining the reg- active. This regulation is well described by the classical

ulation of hemoglobin, enzymes and membrane receptors (2–5). http://www.jimmunol.org/ Monod-Wyman-Changeux model of allostery. More- The ab TCR is an oligomeric protein complex that forms over, the observation that TCRs assemble into nano- higher-order clusters in the plasma membrane of T cells (6). clusters might allow for homotropic allostery, in which Although traditionally the TCR has not been associated with individual TCRs could positively cooperate and thus allostery, recent data strongly suggest it is regulated allosteri- enhance the sensitivity of T cell activation. This new cally. In this Brief Review, we will discuss the evidence sup- view of TCR regulation will contribute to a better porting this idea. understanding of TCR functioning. The Journal of Immunology, 2017, 198: 47–52. Allostery Following the concept introduced by Monod et al. (1), allo- by guest on September 25, 2021 stery is based on the premise that a regulated protein can exist ife depends to a large extent on the precise control of in two different conformations, which can be defined by molecular events. Proteins occupy a central place in changes of the protein’s tertiary and/or quaternary structure. L molecular regulation. In the early 1960s, it became In one conformation, traditionally called the tense state, the clear that different ligands can positively or negatively affect protein is inactive and binds to its ligand with low affinity. In the function of a protein at the same time. This has raised the the other conformation, the relaxed state, the protein is active question of how such inputs are integrated. Moreover, in and binds with high affinity to the ligand. Usually, the tense several cases switch-like responses of the proteins were seen, state represents the resting state of the protein. Upon binding profoundly changing their activity over a relatively small range of the ligand, the protein switches to the relaxed state, the of ligand concentration. The landmark paper by Monod et al. active state. Two different mechanisms have been proposed to (1) first presented an elegant and unified explanation of these explain the molecular mechanism leading to the conforma- phenomena by introducing a quantitative model of allosteric tional switch in oligomeric proteins. regulation; to date, it remains one of the most cited papers of On one hand, the Koshland-Nemethy-Filmer (KNF) molecular biology. Allostery is derived from the Greek words model suggests the ligand binds to the protein in its tense allο (different) and stereο (relating to solid objects and conformation and by an induced-fit mechanism instructs the

*Department of Immunology, Institute for Biology III, Faculty of Biology, University of Spemann Graduate School Programme Grant GSC-4 [to W.W.A.S.], Grant SCHA Freiburg, 79108 Freiburg, Germany; †BIOSS Centre for Biological Signaling Studies, 976/2-1 [to W.W.A.S.], and Grant MI 1942/1 [to S.M.]), as well as European Union– University of Freiburg, 79104 Freiburg, Germany; ‡Center for Chronic Immunodefi- European Research Council 2013-Advanced Grant 334763, Novel Properties of An- ciency, Medical Center, University of Freiburg, Faculty of Medicine, University of tigen Receptors and Instruments to Modulate Lymphoid Function in Physiological x Freiburg, 79106 Freiburg, Germany; Centro de Biologıá Molecular Severo Ochoa, and Pathological Conditions Project (to B.A.). Consejo Superior de Investigaciones Cientı´ficas–Universidad Autońoma de Madrid, { Address correspondence and reprint requests to Prof. Wolfgang W.A. Schamel, Univer- 28049 Madrid, Spain; Division of Theoretical Systems Biology, German Cancer Re- ‖ sity of Freiburg, 79108 Freiburg, Germany. E-mail address: wolfgang.schamel@biologie. search Center, 69120 Heidelberg, Germany; and BioQuant Center, University of uni-freiburg.de Heidelberg, 69120 Heidelberg, Germany Abbreviations used in this article: KNF, Koshland–Nemethy–Filmer; MWC, Monod– Received for publication September 26, 2016. Accepted for publication November 1, Wyman–Changeux; pMHC, peptide–MHC; PRS, proline-rich sequence; wt, wild 2016. type. This work was supported by funds from the Deutsche Forschungsgemeinschaft (Cluster of Excellence Project EXC294 through BIOSS Center for Biological Signaling Studies Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 [to W.W.A.S.], Cellular Networks Cluster of Excellence Project EXC81 [to T.H.],

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601661 48 BRIEF REVIEWS: TCR ALLOSTERY protein to switch to the relaxed conformation (7). Thus, only CD3ed,CD3eg,andCD3zz dimers, which contain intra- those proteins that are ligand-bound switch to the relaxed state. cellular signaling motifs (9–11). These motifs include tyro- On the other hand, the MWC model proposes that the protein sines that are phosphorylated upon pMHC binding (12, 13) can switch spontaneously from the tense to the relaxed state, and a proline-rich sequence in the CD3e tail (CD3e PRS) even in the absence of a ligand, in a process called allosteric that can associate with the adaptor protein Nck (14, 15). As a transition (1). The ligand binds with higher affinity to the prerequisite for allosteric regulation, the TCR exists in (at relaxed conformation of the protein rather than instructing least) two different conformations. High resolution crystal the protein to change its conformation. This is called selection and nuclear magnetic resonance structures of certain isolated of a conformation. According to the MWC model, negative domains of TCRab and CD3 and a low resolution electron allosteric regulators bind to the protein in the tense state and microscopy structure exist (16–18). Unfortunately, these have thus shift the equilibrium to the inactive state. In contrast, not helped generate a three–dimensional, high-resolution positive allosteric regulators bind to the relaxed state and thus structure of the complete TCR complex. Using other meth- stabilize the protein in the active state (Fig. 1). In most ods, ample experimental evidence has been generated for two proteins studied in detail, the MWC model provides an ex- different TCR conformations, which might include quater- cellent fit to the experimental data (2, 3). nary changes within the spatial arrangement of the multi- Moreover, two forms of allostery can be distinguished: het- protein complex. This evidence has been summarized (19, 20) erotropic and homotropic. Heterotropic allostery works with and includes a biochemical effector-binding assay (14), ac- mono- and multimeric proteins. Binding of a regulator to the cessibility of an epitope (21), acquisition of protease resistance Downloaded from protein regulates the binding of another ligand or substrate (22), measurement of the distance between individual sub- (Fig. 1A). This effect is reciprocal for thermodynamic reasons; units within a TCR (23), and determination of lipid binding the binding of the ligand/substrate also affects the binding of to the transmembrane regions of the TCR (24). In addition, it the regulator. Thus, at least two different binding partners was suggested that the cytoplasmic tails of CD3e and CD3z regulate the protein’s function. Homotropic allostery occurs if are aligned to the membrane in resting TCRs and released the protein is an oligomer. The binding of the first ligand from the membrane upon TCR activation (17, 25–27). The http://www.jimmunol.org/ changes the affinity of the protein for the second, chemically molecular mechanism causing dissociation from the mem- identical, ligand (Fig. 1B). According to the MWC model, the brane is unknown and might be part of the allosteric con- conformational change occurs simultaneously for all binding formational changes discussed in this article. sites (or subunits) of an oligomer or for none of them: this is an The two conformations of the TCR are: 1) the resting all-or-none molecular switch. Thus, the conformational tran- conformation, in which the CD3e PRS cannot bind to Nck, sition is concerted and conserves the symmetry of the oligomer the CD3z cytoplasmic tails are separated from each other, (Fig. 1B). More recently, the graded propagation of allosteric cholesterol binds to the transmembrane region of TCRb, and

states has been suggested for large multiprotein complexes (8). the cytoplasmic tyrosines are not phosphorylated; and 2) the by guest on September 25, 2021 active conformation, which is stabilized after multivalent The TCR pMHC binding. In the active conformation, the CD3e PRS Activation of ab T cells initiates an adaptive immune re- is accessible for Nck binding, the CD3z tails are close to each sponse and is mediated by stimulation of the TCR by its other, cholesterol dissociates from TCRb, and the cytoplas- ligand peptide-MHC (pMHC). The TCR comprises the mic tyrosines are accessible to tyrosine kinases (14, 23, 24, 28, variable TCRab dimer, which binds to pMHC, and the 29). The capacity of TCRs to switch to the active conformation is absolutely required for TCR phosphorylation and T cell stimulation. This was shown using artificial ligands (28) and TCR mutants that are trapped in the resting conformation (CD3eC80G and CD3eK76T) (25, 30, 31). To stabilize the active conformation and induce TCR signaling, soluble anti-TCR/CD3 Abs and soluble pMHC need to bind multivalently to the TCRs (28, 32–34). Of note, in Fig. 2 we show pMHC as a dimer, but omitted to show the TCR as a dimer for simplicity. Whether binding of membrane-bound (in contrast to soluble) monomeric pMHC might be sufficient to stabilize the active TCR conformation is as yet unknown. T cells can already be activated when only very few TCRs are stimulated by pMHC (35, 36). In this respect, some important features of TCR biology are still incompletely under- stood. First, how is it guaranteed that in the absence of a FIGURE 1. Hetero- and homotropic allostery. (A) In heterotropic allostery, binding of a negative or positive allosteric regulator (NAR or PAR, respec- ligand most TCRs are in an inactive resting state? Second, tively) reduces or enhances binding of the main ligand compared with the which mechanisms contribute to the extremely high sensi- absence of any regulator. The sequence of events is in accordance with a tivity of T cell activation? Allosteric regulation of the TCR spontaneous cycling of the protein between the tense and relaxed confor- could provide both answers. mations and conformational selection by the allosteric regulators. (B)In homotropic allostery, binding of one ligand would enhance the binding of Heterotropic allostery at the TCR further ligands to the same multiprotein complex. In this scenario, the first ligand is a positive allosteric regulator. The sequence of events is in accordance When we found the first evidence, to our knowledge, in favor with the MWC model of allostery. of conformational changes in the TCR as an outside-in The Journal of Immunology 49

approaches, TCRs became signaling active and led to a low level of T cell stimulation. Hence, these data demonstrate that it is important to keep the resting–active equilibrium shifted toward the resting state to prevent ligand-independent signaling. Furthermore, preventing cholesterol dissociation from the TCR by covalent coupling resulted in the inability of these TCRs to become signaling active (24), enforcing the view that cholesterol allosterically regulates the TCR according to the MWC model. In conclusion, cholesterol is a natural negative allosteric regulator of the TCR that guarantees that in the absence of a ligand most TCRs are in the resting state.

Homotropic allostery at the TCR In effector and memory T cells as well as in T cells lines, the TCR is present in so-called nanoclusters, in which several TCRs cluster together (41–45). The exact nature of the nanocluster arrangement is not known and some studies were unable to detect TCR nanoclusters (46). Whether the TCR Downloaded from nanoclusters are stable and highly organized symmetrical as- semblies, or individual TCRs transiently come together in small regions of the plasma membrane remains unresolved (6, 47). However, the presence of TCR nanoclusters opens the FIGURE 2. The TCR is regulated by heterotropic allostery. (A) According possibility for a further layer of TCR regulation by homo- to the KNF model, multivalent binding of pMHC to TCRs in the resting tropic allostery (Fig. 1B) as previously suggested using the http://www.jimmunol.org/ conformation would instruct the TCR to switch to the active conformation. e e B CD3 C80G or CD3 K76T mutants (30). This model is not in line with the existing experimental data. ( ) According According to the KNF model, within a nanocluster only the to the MWC model, the TCR switches spontaneously from the resting to the active conformation. Because multivalent pMHC would only bind to the TCRs that are engaged multivalently by pMHC would switch TCRs in the active conformation, this state is stabilized. This model is in line to the active conformation (Fig. 3A, left panel). In this sce- with experimental data. (C) Cholesterol is a negative allosteric regulator of the nario, the presence of small amounts of the mutant CD3e TCR, because it only binds to TCRs in the resting conformation, thus sta- molecules (CD3eC80G or CD3eK76T) that cannot adopt bilizing the resting state. K1 and K2 are binding constants and L the allosteric the active conformation would not inhibit signaling by the equilibrium constant. TCRr, TCR in the resting conformation; TCRa, TCR wild type (wt) TCR (Fig. 3A, right panel). However, we by guest on September 25, 2021 in the active conformation. found that expression of 5% mutant CD3e molecules sig- nificantly inhibited signaling by wt TCRs. This strong signaling mechanism, we wrote that “ligand engagement of dominant negative effect of the CD3e mutant suggested the TCR-CD3 induces a conformational change” (14). This existence of allosteric effects among TCRs within nanoclusters postulate could have implied that pMHC binds to TCRs in that adopt either the resting or active conformations in a the resting conformation, then leading the TCR to adopt the coordinated fashion (30). These allosteric effects are again active conformation (Fig. 2A). This scenario would be in line best explained by the MWC model, because in this model the with the KNF model. However, later data demonstrated that presence of small amounts of CD3eC80G or CD3eK76T TCRs spontaneously switch to the active conformation even would inhibit also the wt TCRs to adopt the active confor- in the absence of pMHC (24, 37, 38). Thus, the MWC model mation (Fig. 3B), as seen experimentally (30). seems to better reflect the conformational regulation of the Upon pMHC stimulation, several TCR nanoclusters come TCR: the TCR can switch between two conformations by together into microclusters (48, 49). Single molecule resolu- itself, and multivalent pMHC binds preferentially to the active tion of these TCR microclusters has also suggested the exis- conformation, thus stabilizing this state (Fig. 2B). As a result of tence of positive allostery within TCR nanoclusters. The ligand (pMHC) binding, the equilibrium between the resting tyrosine kinase ZAP-70 binds with its tandem SH2 domains and the active states is shifted toward the active one. to phosphorylated TCRs, but not to resting unphosphory- The spontaneous shift of the TCR between its conforma- lated TCRs (50). A first work using the tyrosine kinase ZAP-70 tions was particularly obvious when cholesterol extraction in as a probe of signaling TCRs showed an uneven distribution of the absence of ligand binding led to the accumulation of active ZAP-70 within TCR microclusters, resulting in concentration TCRs and initiated spontaneous TCR signaling (24, 39, 40). of ZAP-70 in specific areas (51). These data could be inter- Thus, cholesterol heterotropically and negatively regulates the preted in a way that in some nanoclusters all TCRs are in the functioning of the TCR as described by the MWC model: active state and in others all TCRs are in the resting state, and cholesterol only binds to and stabilizes TCRs in the resting these two different nanocluster states coexist within a micro- conformation (Fig. 2C). Preventing cholesterol binding to the cluster. Likewise, the use of phospho-specific anti-CD3z Abs TCR results in a shift of the resting–active state equilibrium has shown the existence of high-density TCR nanoclusters with to the active state as demonstrated by either extracting cho- abundant phosphorylated CD3z and low-density TCR nano- lesterol from the membrane, by oxidizing cholesterol to clusters with a low number of phosphorylated CD3z (52). cholestenone, or by mutating the TCRb transmembrane re- These last data suggest the existence of organized, densely gion so that cholesterol can no longer bind (24). In all these packed TCR nanoclusters, which can allow mechanisms of 50 BRIEF REVIEWS: TCR ALLOSTERY

constant of the unligated TCR (neither cholesterol nor pMHC interactions) was shown to be skewed strongly in favor of the resting state [L = (resting TCR)/([spontaneously] active TCR) = 14] (Fig. 2C). Physiological cholesterol levels enhance the occupancy of the resting state by a further factor of four, showing the quantitative impact of this negative allosteric regulator on the activation threshold. A critical feature of the MWC model is the dependence of ligand affinity on the conformational state of the protein, predicting that the TCR in the resting state should bind pMHC with a lower affinity than TCRs in the active state. In line with this idea, mechanical pulling on the TCR, which might have caused the TCR to switch to the active state, enhanced the TCR’s affinity toward pMHC as measured by a prolonged dwell time of the pMHC-TCR interaction (58). Hence, the databased model of heterotropic TCR allostery will inform further analysis of possible homotropic allosteric effects in TCR nanoclusters. Downloaded from Conclusions In this Brief Review we have presented a new view on the FIGURE 3. The TCR is regulated by homotropic allostery. (A) According functioning of the TCR by taking into account the different to the KNF model, only those TCRs within a nanocluster that are multi- conformations it can adopt. We have discussed ample ex- valently pMHC-bound switch to the active conformation. Thus, the presence

perimental evidence that suggests the TCR is allosterically http://www.jimmunol.org/ e of a CD3 mutant that cannot adopt the active conformation would not regulated, and that the MWC concerted allostery model best inhibit signaling by the wt TCRs. This model is not in line with experimental data. (B) According to the MWC model, all TCRs within a nanocluster are explains these observations. This refers to heterotropic allostery either present in the resting or in the active state. Thus, the CD3e mutant that in which cholesterol serves as a negative allosteric regulator of cannot switch to the active conformation would prevent all TCRs in the the TCR and by homotropic allostery within TCR nano- nanocluster adopting the active state and, thus would inhibit signaling by the clusters in which positive cooperativity was observed. Thus, wt TCRs. This model is in line with experimental data. (C) The MWC model TCRs can sense their environment, such as the concentration proposes positive cooperativity between the TCRs in a nanocluster, implying of cholesterol and pMHC, and respond accordingly. This new that multivalent binding of one pMHC dimer would enhance the binding of view on the TCR suggests ﬁve novel considerations: further pMHC dimers to the same TCR nanocluster. Whether this is the case by guest on September 25, 2021 has still to be experimentally tested. TCRs in the active state are shown in (1) Cholesterol is a negative regulator of the spontaneous pink and those in the resting state in blue. The black TCRs contain the conformational switch of the TCR, reducing TCR sig- mutant CD3e molecule and are trapped in the resting conformation. naling (see the section headed Heterotropic allostery at the TCR). At the same time, cholesterol contributes to allostery versus disorganized, perhaps randomly associated, TCR nanoclustering, because cholesterol extraction TCR clusters. leads to disassembly of the nanoclusters. In this respect Together, these experimental data suggest there is a strong cholesterol is a positive regulator of TCR functioning positive cooperativity between individual TCRs within one (59, 60). Thus, it would be important to study under nanocluster. These nanoclusters might contain up to 20 TCRs which conditions these opposing functions dominate (6, 43–45) and binding of only two pMHC (a pMHC dimer) and which other lipids are involved in TCR regulation. might stabilize all 20 TCRs in the signaling competent state. (2) Homotropic allostery implies that TCRs communicate Hence, this concerted switch to the active conformation within a nanocluster. Whether this is done by protein– might help to explain the high sensitivity of T cell activation. protein interactions (such as CD3z–CD3z contacts) or mediated by lipids and whether there is symmetry in Quantitating TCR allostery the nanoclusters, as often suggested for homotropic The litmus test for any model of allosteric regulation is whether allostery (2), would be important areas of research. it can quantitatively account for experimental data and make Certainly, the highest goal would be to obtain a three- valid predictions for new experiments. This has previously dimensional structure of the TCR and TCR nanoclusters. been achieved for classical examples such as hemoglobin (1, 2, (3) Likewise, structural determination of the resting and 53), phosphofructokinase (54, 55), and aspartate trans- active conformations of the TCR would allow atomic carbamoylase (56). Also, for the transcription factor NFAT, insight into the conformational change and help ex- which is pivotal in T cell activation, experimental data on plain how multivalent pMHC binding stabilizes the multisite phosphorylation have been explained by an allo- active state. Most likely the conserved diagonal orien- steric conformational switch model, where the individual tation of the pMHC–TCRab interaction, in which the phosphorylation sites take the role of the ligand binding sites N terminus of the peptide interacts with TCRa and (57). For the heterotropic regulation of the TCR by cholesterol the C terminus with TCRb, plays a role. This has been and pMHC, Swamy et al. (24) showed the MWC model best elaborated by the permissive geometry model (19). accounted for the experimental data (see Heterotropic allostery (4) Besides the TCR, the coreceptors CD8 and CD4 also at the TCR and Fig. 2). The conformational equilibrium bind to pMHC. Because the coreceptors might also The Journal of Immunology 51

interact with the TCR, it is possible that they directly 20. Blanco, R., and B. Alarcoń. 2012. TCR nanoclusters as the framework for trans- mission of conformational changes and cooperativity. Front. Immunol. 3: 115. impact on the allosteric switch of the TCR (61, 62). 21. Risuenõ, R. M., D. Gil, E. Fernańdez, F. Sańchez-Madrid, and B. Alarcoń. 2005. Furthermore, differential glycosylation of the coreceptors Ligand-induced conformational change in the T-cell receptor associated with pro- ductive immune synapses. Blood 106: 601–608. or the TCR in different developmental or differentiation 22. Risuenõ, R. M., W. W. Schamel, and B. Alarcoń. 2008. T cell receptor engagement stages (63) could alter the TCR’s resting–active equilib- triggers its CD3epsilon and CD3zeta subunits to adopt a compact, locked con- rium and thus influence the sensitivity of T cell activation. formation. PLoS One 3: e1747. 23. Lee, M. S., C. R. Glassman, N. R. Deshpande, H. B. Badgandi, H. L. Parrish, (5) One important consequence of homotropic allostery C. Uttamapinant, P. S. Stawski, A. Y. Ting, and M. S. Kuhns. 2015. A mechanical would be that multivalent pMHC binding to some switch couples T cell receptor triggering to the cytoplasmic juxtamembrane regions of CD3zz. Immunity 43: 227–239. TCRs of a T cell would enhance the binding of the 24. Swamy, M., K. Beck-Garcia, E. Beck-Garcia, F. A. Hartl, A. Morath, O. S. Yousefi, following pMHC (Fig. 3C). This would enhance T cell E. P. Dopfer, E. Molna´r, A. K. Schulze, R. Blanco, et al. 2016. A cholesterol-based allostery model of T cell receptor phosphorylation. Immunity 44: 1091–1101. activation and is an important point to experimentally 25. Dopfer, E. P., F. A. Hartl, H. H. Oberg, G. M. Siegers, O. S. Yousefi, S. Kock, address. Homotropic allostery could also have impor- G. J. Fiala, B. Garcillań, A. Sandstrom, B. Alarcoń, et al. 2014. The CD3 con- tant implications for ligand discrimination by the formational change in the gd T cell receptor is not triggered by antigens but can be enforced to enhance tumor killing. Cell Rep. 7: 1704–1715. TCR. T cells are able to accurately measure the 26. Aivazian, D., and L. J. Stern. 2000. Phosphorylation of T cell receptor z is regulated affinity/avidity and/or the dwell time of a pMHC– by a lipid dependent folding transition. Nat. Struct. Biol. 7: 1023–1026. 27. Shi, X., Y. Bi, W. Yang, X. Guo, Y. Jiang, C. Wan, L. Li, Y. Bai, J. Guo, Y. Wang, TCR interaction and respond accordingly (64–69). et al. 2013. Ca2+ regulates T-cell receptor activation by modulating the charge However, considering TCR nanoclusters, multivalent property of lipids. Nature 493: 111–115. 28. Minguet, S., M. Swamy, B. Alarcoń, I. F. Luescher, and W. W. Schamel. 2007. Full Downloaded from binding of the first pMHC would enhance the avidity activation of the T cell receptor requires both clustering and conformational changes of following pMHC. Thus, allosteric regulation of the at CD3. Immunity 26: 43–54. TCR might not only impact on the sensitivity of a 29. Gil, D., A. G. Schrum, B. Alarcoń, and E. Palmer. 2005. T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 T cell response, but also on the quality. complex of thymocytes. J. Exp. Med. 201: 517–522. 30. Martıńez-Martıń, N., R. M. Risuenõ, A. Morreale, I. Zaldı´var, E. Fernańdez- Arenas, F. Herranz, A. R. Ortiz, and B. Alarcoń. 2009. Cooperativity between T cell We expect that this new view on the TCR will stimulate receptor complexes revealed by conformational mutants of CD3epsilon. Sci. Signal. novel experiments and unravel a number of new mechanisms 2: ra43. http://www.jimmunol.org/ that regulate TCR functioning. 31. Blanco, R., A. Borroto, W. Schamel, P. Pereira, and B. Alarcon. 2014. Confor- mational changes in the T cell receptor differentially determine T cell subset de- velopment in mice. Sci. Signal. 7: ra115. 32. Chang, T. W., P. C. Kung, S. P. Gingras, and G. Goldstein. 1981. Does OKT3 Disclosures monoclonal antibody react with an antigen-recognition structure on human T cells? The authors have no financial conflicts of interest. Proc. Natl. Acad. Sci. USA 78: 1805–1808. 33. Cochran, J. R., T. O. Cameron, and L. J. Stern. 2000. The relationship of MHC- peptide binding and T cell activation probed using chemically defined MHC class II References oligomers. Immunity 12: 241–250. 34. Boniface, J. J., J. D. Rabinowitz, C. Wulfing,€ J. Hampl, Z. Reich, J. D. Altman, 1. Monod, J., J. Wyman, and J. P. Changeux. 1965. On the nature of allosteric R. M. Kantor, C. Beeson, H. M. McConnell, and M. M. Davis. 1998. Initiation of

transitions: a plausible model. J. Mol. Biol. 12: 88–118. by guest on September 25, 2021 signal transduction through the T cell receptor requires the peptide multivalent 2. Changeux, J. P. 2013. 50 years of allosteric interactions: the twists and turns of the engagement of MHC ligands. Immunity 9: 459–466. models. Nat. Rev. Mol. Cell Biol. 14: 819–829. 35. Purbhoo, M. A., D. J. Irvine, J. B. Huppa, and M. M. Davis. 2004. T cell killing 3. Gerhart, J. 2014. From feedback inhibition to allostery: the enduring example of does not require the formation of a stable mature immunological synapse. Nat. aspartate transcarbamoylase. FEBS J. 281: 612–620. Immunol. 5: 524–530. 4. Christopoulos, A. 2014. Advances in G protein-coupled receptor allostery: from 36. Irvine, D. J., M. A. Purbhoo, M. Krogsgaard, and M. M. Davis. 2002. Direct function to structure. Mol. Pharmacol. 86: 463–478. observation of ligand recognition by T cells. Nature 419: 845–849. 5. Edelstein, S. J. 1975. Cooperative interactions of hemoglobin. Annu. Rev. Biochem. 37. de la Cruz, J., T. Kruger, C. A. Parks, R. L. Silge, N. S. van Oers, I. F. Luescher, 44: 209–232. A. G. Schrum, and D. Gil. 2011. Basal and antigen-induced exposure of the ´ 6. Schamel, W. W., and B. Alarcon. 2013. Organization of the resting TCR in proline-rich sequence in CD3e. J. Immunol. 186: 2282–2290. nanoscale oligomers. Immunol. Rev. 251: 13–20. 38. Mingueneau, M., A. Sansoni, C. Gre´goire, R. Roncagalli, E. Aguado, A. Weiss, ´ 7. Koshland, Jr., D. E., G. Nemethy, and D. Filmer. 1966. Comparison of experi- M. Malissen, and B. Malissen. 2008. The proline-rich sequence of CD3epsilon mental binding data and theoretical models in proteins containing subunits. Bio- controls T cell antigen receptor expression on and signaling potency in preselection chemistry 5: 365–385. CD4+CD8+ thymocytes. Nat. Immunol. 9: 522–532. 8. Bray, D. 2013. The propagation of allosteric states in large multiprotein complexes. 39. Rouquette-Jazdanian, A. K., C. Pelassy, J. P. Breittmayer, and C. Aussel. 2006. J. Mol. Biol. 425: 1410–1414. Revaluation of the role of cholesterol in stabilizing rafts implicated in T cell receptor 9. Guy, C. S., and D. A. Vignali. 2009. Organization of proximal signal initiation at signaling. Cell. Signal. 18: 105–122. the TCR:CD3 complex. Immunol. Rev. 232: 7–21. 40. Kabouridis, P. S., J. Janzen, A. L. Magee, and S. C. Ley. 2000. Cholesterol de- 10. Alarcoń, B., D. Gil, P. Delgado, and W. W. Schamel. 2003. Initiation of TCR pletion disrupts lipid rafts and modulates the activity of multiple signaling pathways signaling: regulation within CD3 dimers. Immunol. Rev. 191: 38–46. in T lymphocytes. Eur. J. Immunol. 30: 954–963. 11. Kuhns, M. S., and M. M. Davis. 2012. TCR signaling emerges from the sum of 41. Fernańdez-Miguel, G., B. Alarcoń, A. Iglesias, H. Bluethmann, M. Alvarez-Mon, many parts. Front. Immunol. 3: 159. E. Sanz, and A. de la Hera. 1999. Multivalent structure of an alphabetaT cell re- 12. Reth, M. 1989. Antigen receptor tail clue. Nature 338: 383–384. ceptor. Proc. Natl. Acad. Sci. USA 96: 1547–1552. 13. Weiss, A. 2010. The right team at the right time to go for a home run: tyrosine 42. Schamel, W. W., I. Arechaga, R. M. Risuenõ, H. M. van Santen, P. Cabezas, kinase activation by the TCR. Nat. Immunol. 11: 101–104. C. Risco, J. M. Valpuesta, and B. Alarcoń. 2005. Coexistence of multivalent and 14. Gil, D., W. W. Schamel, M. Montoya, F. Sańchez-Madrid, and B. Alarcoń. 2002. monovalent TCRs explains high sensitivity and wide range of response. J. Exp. Med. Recruitment of Nck by CD3 epsilon reveals a ligand-induced conformational change 202: 493–503. essential for T cell receptor signaling and synapse formation. Cell 109: 901–912. 43. Kumar, R., M. Ferez, M. Swamy, I. Arechaga, M. T. Rejas, J. M. Valpuesta, 15. Kesti, T., A. Ruppelt, J. H. Wang, M. Liss, R. Wagner, K. Taskeń, and K. Saksela. W. W. Schamel, B. Alarcon, and H. M. van Santen. 2011. Increased sensitivity of 2007. Reciprocal regulation of SH3 and SH2 domain binding via tyrosine phos- antigen-experienced T cells through the enrichment of oligomeric T cell receptor phorylation of a common site in CD3epsilon. J. Immunol. 179: 878–885. complexes. Immunity 35: 375–387. 16. Kuhns, M. S., M. M. Davis, and K. C. Garcia. 2006. Deconstructing the form and 44. Lillemeier, B. F., M. A. Mo¨rtelmaier, M. B. Forstner, J. B. Huppa, J. T. Groves, function of the TCR/CD3 complex. Immunity 24: 133–139. and M. M. Davis. 2010. TCR and Lat are expressed on separate protein islands on 17. Xu, C., E. Gagnon, M. E. Call, J. R. Schnell, C. D. Schwieters, C. V. Carman, T cell membranes and concatenate during activation. Nat. Immunol. 11: 90–96. J. J. Chou, and K. W. Wucherpfennig. 2008. Regulation of T cell receptor acti- 45. Sherman, E., V. Barr, S. Manley, G. Patterson, L. Balagopalan, I. Akpan, vation by dynamic membrane binding of the CD3e cytoplasmic tyrosine-based C. K. Regan, R. K. Merrill, C. L. Sommers, J. Lippincott-Schwartz, and motif. Cell 135: 702–713. L. E. Samelson. 2011. Functional nanoscale organization of signaling molecules 18. Arechaga, I., M. Swamy, D. Abia, W. A. Schamel, B. Alarcoń, and J. M. Valpuesta. downstream of the T cell antigen receptor. Immunity 35: 705–720. 2010. Structural characterization of the TCR complex by electron microscopy. Int. 46. James, J. R., J. McColl, M. I. Oliveira, P. D. Dunne, E. Huang, A. Jansson, Immunol. 22: 897–903. P. Nilsson, D. L. Sleep, C. M. Gonçalves, S. H. Morgan, et al. 2011. The T cell 19. Minguet, S., and W. W. A. Schamel. 2008. A permissive geometry model for TCR- receptor triggering apparatus is composed of monovalent or monomeric proteins. J. CD3 activation. Trends Biochem. Sci. 33: 51–57. Biol. Chem. 286: 31993–32001. 52 BRIEF REVIEWS: TCR ALLOSTERY

47. Alarcoń, B., M. Swamy, H. M. van Santen, and W. W. A. Schamel. 2006. T-cell 59. Wang, F., K. Beck-Garcıá, C. Zorzin, W. W. Schamel, and M. M. Davis. 2016. antigen-receptor stoichiometry: pre-clustering for sensitivity. EMBO Rep. 7: 490–495. Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring 48. Varma, R., G. Campi, T. Yokosuka, T. Saito, and M. L. Dustin. 2006. T cell derivative of membrane cholesterol. Nat. Immunol. 17: 844–850. receptor-proximal signals are sustained in peripheral microclusters and terminated in 60. Molna´r, E., M. Swamy, M. Holzer, K. Beck-Garcıá, R. Worch, C. Thiele, the central supramolecular activation cluster. Immunity 25: 117–127. G. Guigas, K. Boye, I. F. Luescher, P. Schwille, et al. 2012. Cholesterol and 49. Yokosuka, T., K. Sakata-Sogawa, W. Kobayashi, M. Hiroshima, A. Hashimoto- sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering. J. Tane, M. Tokunaga, M. L. Dustin, and T. Saito. 2005. Newly generated T cell Biol. Chem. 287: 42664–42674. 61. Gil, D., A. G. Schrum, M. A. Daniels, and E. Palmer. 2008. A role for CD8 in the receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 developmental tuning of antigen recognition and CD3 conformational change. J. and SLP-76. Nat. Immunol. 6: 1253–1262. Immunol. 180: 3900–3909. 50. Chan, A. C., M. Iwashima, C. W. Turck, and A. Weiss. 1992. ZAP-70: a 70 kd 62. Yoon, S. T., U. Dianzani, K. Bottomly, and C. A. J. Janeway, Jr. 1994. Both high protein-tyrosine kinase that associates with the TCR zeta chain. Cell 71: 649–662. and low avidity antibodies to the T cell receptor can have agonist or antagonist 51. Neve-Oz, Y., Y. Razvag, J. Sajman, and E. Sherman. 2015. Mechanisms of localized activity. Immunity 1: 563–569. activation of the T cell antigen receptor inside clusters. Biochim. Biophys. Acta 1853: 63. Daniels, M. A., L. Devine, J. D. Miller, J. M. Moser, A. E. Lukacher, J. D. Altman, 810–821. P. Kavathas, K. A. Hogquist, and S. C. Jameson. 2001. CD8 binding to MHC class 52. Pageon, S. V., T. Tabarin, Y. Yamamoto, Y. Ma, J. S. Bridgeman, A. Cohnen, I molecules is influenced by T cell maturation and glycosylation. Immunity 15: C. Benzing, Y. Gao, M. D. Crowther, K. Tungatt, et al. 2016. Functional role of 1051–1061. T-cell receptor nanoclusters in signal initiation and antigen discrimination. Proc. 64. Daniels, M. A., E. Teixeiro, J. Gill, B. Hausmann, D. Roubaty, K. Holmberg, Natl. Acad. Sci. USA 113: E5454–E5463. G. Werlen, G. A. Holla¨nder, N. R. Gascoigne, and E. Palmer. 2006. Thymic se- 53. Bellelli, A. 2010. Hemoglobin and cooperativity: experiments and theories. Curr. lection threshold defined by compartmentalization of Ras/MAPK signalling. Nature Protein Pept. Sci. 11: 2–36. 444: 724–729. 54. Goldbeter, A. 1973. Patterns of spatiotemporal organization in an allosteric enzyme 65. Aleksic, M., O. Dushek, H. Zhang, E. Shenderov, J. L. Chen, V. Cerundolo, model. Proc. Natl. Acad. Sci. USA 70: 3255–3259. D. Coombs, and P. A. van der Merwe. 2010. Dependence of T cell antigen recog- 55. Otto, M., R. Heinrich, B. Kuhn, and G. Jacobasch. 1974. A mathematical model for nition on T cell receptor-peptide MHC confinement time. Immunity 32: 163–174. 66. Holler, P. D., and D. M. Kranz. 2003. Quantitative analysis of the contribution of

the influence of fructose 6-phosphate, ATP, potassium, ammonium and magnesium Downloaded from TCR/pepMHC affinity and CD8 to T cell activation. Immunity 18: 255–264. on the phosphofructokinase from rat erythrocytes. Eur. J. Biochem. 49: 169–178. 67. Kalergis, A. M., N. Boucheron, M. A. Doucey, E. Palmieri, E. C. Goyarts, Z. Vegh, 56. Howlett, G. J., M. N. Blackburn, J. G. Compton, and H. K. Schachman. 1977. I. F. Luescher, and S. G. Nathenson. 2001. Efficient T cell activation requires an Allosteric regulation of aspartate transcarbamoylase. Analysis of the structural and optimal dwell-time of interaction between the TCR and the pMHC complex. Nat. functional behavior in terms of a two-state model. Biochemistry 16: 5091–5100. Immunol. 2: 229–234. 57. Salazar, C., and T. Ho¨fer. 2003. Allosteric regulation of the transcription factor 68. Laugel, B., H. A. van den Berg, E. Gostick, D. K. Cole, L. Wooldridge, J. Boulter, NFAT1 by multiple phosphorylation sites: a mathematical analysis. J. Mol. Biol. A. Milicic, D. A. Price, and A. K. Sewell. 2007. Different T cell receptor affinity 327: 31–45. thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte acti- 58. Liu, B., W. Chen, B. D. Evavold, and C. Zhu. 2014. Accumulation of dynamic vation and tetramer binding properties. J. Biol. Chem. 282: 23799–23810. http://www.jimmunol.org/ catch bonds between TCR and agonist peptide-MHC triggers T cell signaling. Cell 69. Altan-Bonnet, G., and R. N. Germain. 2005. Modeling T cell antigen discrimi- 157: 357–368. nation based on feedback control of digital ERK responses. PLoS Biol. 3: e356. by guest on September 25, 2021