An evolutionary divergent thermodynamic brake in ZAP-70 fine-tunes the kinetic proofreading in T cells
2022; Elsevier BV; Volume: 298; Issue: 10 Linguagem: Inglês
10.1016/j.jbc.2022.102376
ISSN1083-351X
AutoresKaustav Gangopadhyay, Arnab Roy, Athira C. Chandradasan, Swarnendu Roy, Olivia Debnath, Soumee SenGupta, Subhankar Chowdhury, Dipjyoti Das, Rahul Das,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoT cell signaling starts with assembling several tyrosine kinases and adapter proteins to the T cell receptor (TCR), following the antigen binding to the TCR. The stability of the TCR–antigen complex and the delay between the recruitment and activation of each kinase determines the T cell response. Integration of such delays constitutes a kinetic proofreading mechanism to regulate T cell response to the antigen binding. However, the mechanism of these delays is not fully understood. Combining biochemical experiments and kinetic modeling, here we report a thermodynamic brake in the regulatory module of the tyrosine kinase ZAP-70, which determines the ligand selectivity, and may delay the ZAP-70 activation upon antigen binding to TCR. The regulatory module of ZAP-70 comprises of a tandem SH2 domain that binds to its ligand, doubly-phosphorylated ITAM peptide (ITAM-Y2P), in two kinetic steps: a fast step and a slow step. We show the initial encounter complex formation between the ITAM-Y2P and tandem SH2 domain follows a fast-kinetic step, whereas the conformational transition to the holo-state follows a slow-kinetic step. We further observed a thermodynamic penalty imposed during the second phosphate-binding event reduces the rate of structural transition to the holo-state. Phylogenetic analysis revealed the evolution of the thermodynamic brake coincides with the divergence of the adaptive immune system to the cell-mediated and humoral responses. In addition, the paralogous kinase Syk expressed in B cells does not possess such a functional thermodynamic brake, which may explain the higher basal activation and lack of ligand selectivity in Syk. T cell signaling starts with assembling several tyrosine kinases and adapter proteins to the T cell receptor (TCR), following the antigen binding to the TCR. The stability of the TCR–antigen complex and the delay between the recruitment and activation of each kinase determines the T cell response. Integration of such delays constitutes a kinetic proofreading mechanism to regulate T cell response to the antigen binding. However, the mechanism of these delays is not fully understood. Combining biochemical experiments and kinetic modeling, here we report a thermodynamic brake in the regulatory module of the tyrosine kinase ZAP-70, which determines the ligand selectivity, and may delay the ZAP-70 activation upon antigen binding to TCR. The regulatory module of ZAP-70 comprises of a tandem SH2 domain that binds to its ligand, doubly-phosphorylated ITAM peptide (ITAM-Y2P), in two kinetic steps: a fast step and a slow step. We show the initial encounter complex formation between the ITAM-Y2P and tandem SH2 domain follows a fast-kinetic step, whereas the conformational transition to the holo-state follows a slow-kinetic step. We further observed a thermodynamic penalty imposed during the second phosphate-binding event reduces the rate of structural transition to the holo-state. Phylogenetic analysis revealed the evolution of the thermodynamic brake coincides with the divergence of the adaptive immune system to the cell-mediated and humoral responses. In addition, the paralogous kinase Syk expressed in B cells does not possess such a functional thermodynamic brake, which may explain the higher basal activation and lack of ligand selectivity in Syk. The activation and quiescence in the cell-mediated immune response by T cell is regulated by a kinetic proofreading mechanism (1McKeithan T.W. Kinetic proofreading in T-cell receptor signal transduction.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5042-5046Crossref PubMed Scopus (707) Google Scholar, 2Rabinowitz J.D. Beeson C. Lyons D.S. Davis M.M. McConnell H.M. Kinetic discrimination in T-cell activation.Proc. Natl. Acad. Sci. U. S. 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Mechanisms determining a differential threshold for sensing Src family kinase activity by B and T cell antigen receptors.J. Biol. Chem. 2020; 295: 12935-12945Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 12Sadras T. Martin M. Kume K. Robinson M.E. Saravanakumar S. Lenz G. et al.Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer.Mol. Cell. 2021; 81: 2094-2111.e2099Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). The Syk family of nonreceptor tyrosine kinases, ZAP-70 and Syk, are indispensable in the early stage of TCR and BCR signaling, respectively (10Mocsai A. Ruland J. Tybulewicz V.L. The SYK tyrosine kinase: a crucial player in diverse biological functions.Nat. Rev. Immunol. 2010; 10: 387-402Crossref PubMed Scopus (845) Google Scholar, 13Au-Yeung B.B. Shah N.H. Shen L. Weiss A. ZAP-70 in signaling, biology, and disease.Annu. Rev. Immunol. 2018; 36: 127-156Crossref PubMed Scopus (60) Google Scholar). Both the kinases are activated by recruiting to the membrane following antigen binding (Fig. S1A). The dwell time of the kinases at the membrane determines their response (14Katz Z.B. Novotna L. Blount A. Lillemeier B.F. A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli.Nat. Immunol. 2017; 18: 86-95Crossref PubMed Scopus (53) Google Scholar). ZAP-70 and Syk, both shares a modular structure composed of an N-terminal regulatory module and a C-terminal kinase domain (Fig. 1A) (10Mocsai A. Ruland J. Tybulewicz V.L. The SYK tyrosine kinase: a crucial player in diverse biological functions.Nat. Rev. Immunol. 2010; 10: 387-402Crossref PubMed Scopus (845) Google Scholar, 13Au-Yeung B.B. Shah N.H. Shen L. Weiss A. ZAP-70 in signaling, biology, and disease.Annu. Rev. Immunol. 2018; 36: 127-156Crossref PubMed Scopus (60) Google Scholar). The regulatory module is made up of tandem Src homology 2 (tSH2) domains connected by a helical linker called interdomain A (Fig. 1, A and C). In the inactive state (apo-state), the two SH2 domains adopt an 'L'-like open conformation making them incompatible to ligand binding (Fig. 1C) (15Deindl S. Kadlecek T.A. Brdicka T. Cao X. Weiss A. Kuriyan J. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70.Cell. 2007; 129: 735-746Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 16Gradler U. Schwarz D. Dresing V. Musil D. Bomke J. Frech M. et al.Structural and biophysical characterization of the Syk activation switch.J. Mol. Biol. 2013; 425: 309-333Crossref PubMed Scopus (55) Google Scholar). The Syk kinases are activated by binding to the doubly-phosphorylated immunoreceptor tyrosine based activation motif (ITAM-Y2P) motifs at the TCR or BCR, respectively, through the tSH2 domain (Figs. 1B and S1A) (17Bu J.Y. Shaw A.S. Chan A.C. 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A. 2003; 100: 14828-14833Crossref PubMed Scopus (38) Google Scholar, 35Isakov N. Wange R.L. Burgess W.H. Watts J.D. Aebersold R. Samelson L.E. ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity.J. Exp. Med. 1995; 181: 375-380Crossref PubMed Scopus (170) Google Scholar, 36Grucza R.A. Bradshaw J.M. Mitaxov V. Waksman G. Role of electrostatic interactions in SH2 domain recognition: salt-dependence of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the syk kinase and the single SH2 domain of the src kinase.Biochemistry. 2000; 39: 10072-10081Crossref PubMed Scopus (50) Google Scholar). The mechanism and functional significance of biphasic ligand binding for T cell signaling remains unclear. We present a kinetic model from a comparative study of the tSH2 domain of ZAP-70 and Syk that explains the differential ligand binding. We observed that the tSH2 domain of ZAP-70 binds to ITAM-Y2P in two-step kinetics, fast and slow, compared to one-step binding in Syk. The slow binding to the ZAP-70 tSH2 domain arises from a thermodynamic penalty (brake) that determines the ligand selectivity and biases the conformational equilibrium of the apo-tSH2 domain toward the open conformation. Conversely, such thermodynamic break is nonfunctional in Syk tSH2-domain. Phylogenetic mapping shows that the emergence of the thermodynamic brake coincides with the evolution of the BCR-TCR-MHC like immune system at the divergence of jawless and jawed fish approximately 500 million years ago (37Flajnik M.F. Kasahara M. Origin and evolution of the adaptive immune system: genetic events and selective pressures.Nat. Rev. Genet. 2010; 11: 47-59Crossref PubMed Scopus (547) Google Scholar). The ZAP-70 tSH2 domain binding to the doubly phosphorylated ITAM-ζ1 peptide (ITAM-Y2P-ζ1) produces a biphasic curve with three distinct dissociation constants, Kd1, Kd2, and Kd1∗ (Fig. 1, B–D) (33Gangopadhyay K. Manna B. Roy S. Kumari S. Debnath O. Chowdhury S. et al.An allosteric hot spot in the tandem-SH2 domain of ZAP-70 regulates T-cell signaling.Biochem. J. 2020; 477: 1287-1308Crossref PubMed Scopus (7) Google Scholar). First, the N-terminal phosphotyrosine residue from ITAM-Y2P binds uncooperatively to the C-SH2 phosphate-binding pocket (PBP) with a low nanomolar affinity (Kd1) to form an encounter complex (Fig. 1, C–E). The formation of the tSH2:ITAM-Y2P encounter complex allows the assembly of the N-SH2 PBP. Subsequently, C-terminal phosphotyrosine residue from ITAM-Y2P binds weakly to the newly formed PBP with micromolar affinity (Kd2). In the steady-state, the two binding events are interlinked by a plateau (Fig. 1D). The second binding event remodels the C-SH2 PBP to an intermediate-binding pocket (Kd1∗) producing a hill-coefficient of 3.4 ± 0.36 (suggesting cooperative binding). It was reported previously, the tSH2-domain of ZAP-70 displays hierarchical preference in binding to different ITAM sequences (17Bu J.Y. Shaw A.S. Chan A.C. Analysis of the interaction of ZAP-70 and Syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5106-5110Crossref PubMed Scopus (118) Google Scholar, 35Isakov N. Wange R.L. Burgess W.H. Watts J.D. Aebersold R. Samelson L.E. ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity.J. Exp. Med. 1995; 181: 375-380Crossref PubMed Scopus (170) Google Scholar, 38Labadia M.E. Ingraham R.H. Schembri-King J. Morelock M.M. Jakes S. Binding affinities of the SH2 domains of ZAP-70, p56lck and Shc to the zeta chain ITAMs of the T-cell receptor determined by surface plasmon resonance.J. Leukoc. Biol. 1996; 59: 740-746Crossref PubMed Scopus (26) Google Scholar, 39Zenner G. Vorherr T. Mustelin T. Burn P. Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR-zeta chain.J. Cell Biochem. 1996; 63: 94-103Crossref PubMed Scopus (38) Google Scholar, 40Osman N. Turner H. Lucas S. Reif K. Cantrell D.A. The protein interactions of the immunoglobulin receptor family tyrosine-based activation motifs present in the T cell receptor zeta subunits and the CD3 gamma, delta and epsilon chains.Eur. J. Immunol. 1996; 26: 1063-1068Crossref PubMed Scopus (91) Google Scholar). We begin by asking which part of the biphasic binding isotherm, in the steady-state, is sensitive to the ITAM peptide sequence (Fig. 1B). We comparatively studied the binding of ITAM-Y2P-ζ3 to the tSH2 domain by intrinsic tryptophan fluorescence spectroscopy and isothermal titration calorimetry (ITC) (Figs. 1, B, D–E and S1, B–F). We probed Kd1 and Kd1∗ by fluorescence spectroscopy and Kd2 and Kd1∗ by ITC. To probe the Kd2, we used tSH2R190A mutant that impairs phosphotyrosine binding to C-SH2 PBP (Figs. 1E and S1F). We overserved that ITAM-Y2P-ζ3 binds weakly to the ZAP-70 tSH2 domain, compared to ITAM-Y2P-ζ1, consistent with the previous reports (17Bu J.Y. Shaw A.S. Chan A.C. Analysis of the interaction of ZAP-70 and Syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5106-5110Crossref PubMed Scopus (118) Google Scholar, 41Love P.E. Hayes S.M. ITAM-mediated signaling by the T-cell antigen receptor.Cold Spring Harb. Perspect. Biol. 2010; 2: a002485Crossref PubMed Scopus (112) Google Scholar) (Fig. 1E). Our data revealed that the C-SH2 domain does not distinguish between the two ITAM-Y2P while forming the encounter complex. Both the peptides, ITAM-Y2P-ζ1 and ITAM-Y2P-ζ3, bind to the C-SH2 domain with low nanomolar affinity (Kd1) of 3.3 ± 0.5 nM and 6.8 ± 1.5 nM, respectively. However, we noted a significant increase in the plateau width for the ITAM-Y2P-ζ3 and tSH2 interaction (Fig. 1D). The ITAM-Y2P-ζ3 binding perturbed the Kd2 and Kd1∗ contributing to the overall increase in the dissociation constant (Fig. 1E). We ask why the plateau-width in the steady-state binding (Fig. 1D) is sensitive to the subtle changes in ligand type? We developed a multistep mathematical-kinetic model to explain the biphasic bindings of ITAM-Y2P and tSH2 domains. This model comprised of different tSH2 domain conformations, open and closed, connected by a complex network (Fig. 2, A and B). The tSH2-apo state (Ropen00) ultimately reaches the tSH2-holo state (Rclosed11) through different pathways associated with distinct rates. The receptor in the apo-state first converts to an encounter complex (Ropen01) and then adopts a closed conformation (Rclosed01). We also considered two other intermediates (Rclosed00 and Rclosed10) through which the final holo-state may form (see Experimental procedures). Based on our experimental dissociation constants, we assumed that the formation of the encounter complex is the fastest (Kd1=kbkf=3−10nM; Fig. 1E). To explain the biphasic binding (Fig. 1D), we further assumed that the transitions to the holo-state from the partially bound states (from Rclosed10 or Rclosed01 to Rclosed11) exhibit negative cooperativity. These steps represent kinetic penalties (42Sevlever F. Di Bella J.P. Ventura A.C. Discriminating between negative cooperativity and ligand binding to independent sites using pre-equilibrium properties of binding curves.PLoS Comput. Biol. 2020; 16e1007929Crossref PubMed Scopus (4) Google Scholar) and occur with much slower forward rates (w1kf and w2kf, with 0<wi<1 , i=1 or 2) with dissociation constants Kd2(=kb1w1kf) and Kd1∗ (= kb2w2kf), respectively. It may be noted that our model does not have any feedback regulation (43Das J. Ho M. Zikherman J. Govern C. Yang M. Weiss A. et al.Digital signaling and hysteresis characterize ras activation in lymphoid cells.Cell. 2009; 136: 337-351Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 44Angeli D. Ferrell Jr., J.E. Sontag E.D. Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1822-1827Crossref PubMed Scopus (751) Google Scholar) and is based on allosteric interaction between the two SH2 domains (33Gangopadhyay K. Manna B. Roy S. Kumari S. Debnath O. Chowdhury S. et al.An allosteric hot spot in the tandem-SH2 domain of ZAP-70 regulates T-cell signaling.Biochem. J. 2020; 477: 1287-1308Crossref PubMed Scopus (7) Google Scholar). We anticipate the variation in Kd1, Kd2, or Kd1∗ may determine the steady-state response. The model was analyzed numerically to calculate the ligand-bound fraction (Equations S1 and S2) and to predict the steady-state response and the kinetic behavior. In the steady-state, the Kd1 determines the receptor sensitivity (initial rising slopes of the bound fractions) in low ligand concentration regime (nM level) and also modulates the plateau width (Fig. 2C). The kinetic behavior mostly showed a single exponential decay except in a narrow intermediate range of Kd1 (around 8 nM – 20 nM), where a two-step decay was observed (Figs. 2, D and E and S2, A and B). Next, we assessed the effect of Kd2 (Fig. 1C) variation on the steady-state behavior by changing the penalty factor, w1 (associated with the transition from Rclosed01 to Rclosed11, Fig. 2B). The Kd2 variation modulated the ligand selectivity by altering the plateau width in the steady-state (Fig. 2F). With a higher penalty (equivalently lower w1 or higher Kd2), the plateau width became broader and displayed two-step kinetics with a sharp initial decay and a slow subsequent decrease of the unbound fraction. (Fig. 2, G and H). We asked, does a biphasic behavior in the steady-state arise due to slow transition of the partially-bound closed states (Rclosed01 or Rclosed10) to an open configuration. In our model, we introduced slow transition from Rclosed01 or Rclosed10 states to open configurations (Ropen01 or Ropen00,respectively) by varying the respective rates, at the same time without putting penalties on other steps (Kd2 and Kd1∗). None of the changes made produced any biphasic response in the steady-state (Fig. S2, E–H). In contrast, when we introduce slow transition from Rclosed01 or Rclosed10 to the final holo-state (Rclosed11), the steady-state responses become biphasic (Fig. S2, I–L). Thus, we conclude that the slow transitions (due to the penalty) from the Rclosed01 and Rclosed10 to the Rclosed11 determines the plateau behavior and not the slow relaxation of the close states to the open state. Since both the slow transition from the Rclosed01 or Rclosed10 states to the final holo-state (Rclosed11) could, in principle, lead to a biphasic response, we next asked which transition is more sensitive. We found that the variation of Kd1∗ marginally alters the plateau width in the steady-state (Fig. S2, C and D) (when we introduced penalties in the transitions from Rclosed10 and Rclosed01 to Rclosed11). Therefore, the slowest binding step to the N-SH2 PBP (Kd2) mainly controls the plateau width. This conclusion correlates with the observed Kd2, which is orders of magnitude lower than the Kd1 and Kd1∗ (Fig. 1E), suggesting the corresponding step may impart a significant penalty in the dynamic binding of the ligand. However, measurement of free energy change is necessary to determine if a thermodynamic cost manifests as a kinetic penalty, as elucidated in the model. Finally, for a particular set of parameter choices, the model prediction reasonably agreed with the experimental data of ITAM-Y2P-ζ1 and ITAM-Y2P-ζ3 bindings to the tSH2 domain (Fig. 2I). In the model, reported values of Kd1, Kd2, and Kd1∗ (Fig. 1E) were used for the quantitative matching, but other parameters were unknown and chosen arbitrarily to fit the data (Table S5). Since our model predicted two-step binding kinetics (Fig. 2, E and H), we next probed the binding kinetics of ITAM-Y2P-ζ1 or ITAM-Y2P-ζ3 to the tSH2 domain by stopped-flow fluorescence spectroscopy. We started with mixing excess ITAM-Y2P-ζ1 to the tSH2 domains at 10 °C and measured the change in tryptophan fluorescence intensity for 200 s (Fig. 3A). We observed that the fluorescence intensity decay in two steps, fast ( 20 s) (Fig. 3A). Hence, we recorded all the kinetic experiments at two-time scales. All kinetic data were first normalized against the highest intensity and then subtracted by the blank (protein only sample) (Fig. 3, A and B) and fitted to a one-site association kinetics (Table 1).Table 1Observed rate for the ZAP-70 tSH2 domain and ITAM-Y2P bindingConstructkobsfast (s-1)kobsslow (s-1)tSH2:ITAM-Y2P-ζ123.87 ± 1.010.262 ± 0.0977tSH2:ITAM-Y2P-ζ321.44 ± 1.830.012 ± 0.0048tSH2 R39A:ITAM-Y2P-ζ124.82 ± 2.29---tSH2:ITAM-YP-ζ128.60 ± 5.84---tSH2: ITAM-Y2P-ζ1 E13A24.4 ± 2.040.022 ± 0.0045tSH2 F117A: ITAM-Y2P-ζ121.83 ± 1.490.0517 ± 0.0062tSH2 R43P: ITAM-Y2P-ζ127.96 ± 3.452.25 ± 0.032tSH2 R192A: ITAM-Y2P-ζ1---0.006 ± 0.002 Open table in a new tab We observed that the ITAM-Y2P-ζ1 binds to the tSH2 domain with two observed rates of kobsfast=23.87±1.01 s−1 and kobsslow=0.262±0.098 s−1 (Fig. 3, A, B and Table 1). Our mathematical model suggests that the fast-binding kinetic may arise (Fig. 2, E and H) during the formation of the encounter complex (Kd1). To test that, we turn to three samples, two mutants tSH2R39A and tSH2R192A that would prevent phosphotyrosine binding to N-SH2 and C-SH2 PBP, respectively. Third, a single phosphotyrosine ITAM-ζ1 peptide (ITAM-YP-ζ1) that will show only one binding event (Fig. 3C). The steady-state fluorescence titration of tSH2R39A to ITAM-Y2P-ζ1 and tSH2 domain to ITAM-YP-ζ1 showed that the first binding step is preserved with (Kd1) of 8 ± 1.05 nM and 3.7 ± 0.1 nM, respectively, and no subsequent binding was observed (Figs. 3C, S3, A and B). In the kinetics experiment, both the samples showed a kobsfast of 24.82 ± 2.29 s−1 and 28.60 ± 5.84 s−1, respectively, with no detectable slow binding (Fig. 3, D and E). Under substoichiometric ligand concentration, tSH2R192A did not bind to the ITAM-Y2P-ζ1(33) and showed a linear ligand binding at a higher ITAM-Y2P-ζ1 (μM) concentration (Fig. S3G). The tSH2R192A binds with a ten-fold slower kobsslow (0.006 ± 0.002 s−1) rate in comparison to the WT tSH2 domain (Table 1), with no detectable fast binding (Fig. 3, D and E). Together, our data indicates that the N-SH2 (R39A) and C-SH2 (R192A) mutants display only μM and nM affinity, respectively, suggesting that the formation of the encounter complex is critical for the subsequent ligand binding. To check if our proposed model (Fig. 2B) could explain the above data (Fig. 3, C–E), we introduced a modification in the model (Fig. 3F). The N-SH2 binding is almost absent in both cases, and the partially bound state does not transform to the
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