It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.
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The authors thank R.A. Cornall, M.L. Dustin and P.A. van der Merwe for comments on the manuscript and S. Ikemizu for useful discussions about the structure. We also thank W. Lu and T. Walter for technical support with protein expression and crystallization, the staff at Diamond Light Source beamlines I02, I03 and I04-1 (proposal mx10627) and European Synchrotron Radiation Facility beamlines ID23EH1 and ID23EH2 for assistance at the synchrotrons, G. Sutton for assistance with MALS experiments, and M. Fritzsche for advice on the calcium analysis. This work was funded by the Wellcome Trust (098274/Z/12/Z to S.J.D.; 090532/Z/09/Z to R.J.C.G.; 090708/Z/09/Z to D.K.), the UK Medical Research Council (G0700232 to A.R.A.), the Royal Society (UF120277 to S.F.L.) and Cancer Research UK (C20724/A14414 to C.S.; C375/A10976 to E.Y.J.). The Oxford Division of Structural Biology is part of the Wellcome Trust Centre for Human Genetics, Wellcome Trust Core Award Grant Number 090532/Z/09/Z. We acknowledge financial support from Instruct, an ESFRI Landmark Project. The OPIC electron microscopy facility was funded by a Wellcome Trust JIF award (060208/Z/00/Z).
The authors declare no competing financial interests.
Integrated supplementary information
(a) (Left) N-glycosylated proteins analyzed using MALS (lanes 1–3), cryo-EM (lane 4) and VA-TIRF (lanes 5–7): CD45RABC (lane 1), CD45R0 (lane 2), CD45d1–d4 (lane 3) and Sema6A-CD45R0 (lane 4), and N-terminally HaloTag-tagged forms of CD45RABC (lane 5), CD45R0 (lane 6) and CD45d1–d4 (lane 7). Top and bottom panels show 10% non-reducing (NR) and reducing (R) SDS-PAGE gels, respectively. (Right) Endo F1-deglycosylated proteins used for growing crystals: CD45d1–d4 (lane 1), CD45d1–d3 (lane 2), CD45d1d2 (lane 3) and rCD45d3d4 (lane 4). Top and bottom panels show 15% NR and R SDS-PAGE gels, respectively. (b) Electron density maps for CD45d1d2. (i) KPtCl4-SAD phased, phase-extended and density modified map from SHARP (calculated to 2.3 Å and contoured at 1.1 σ). The two CD45d1d2 molecules in the crystallographic asymmetric unit are colored yellow and magenta, respectively. (ii–iii) Close-up view of the density for the residual GlcNAc (remaining following Endo F1 cleavage of N-linked oligosaccharides) at residue N378. In (ii), a region of the experimental map in (i) is shown. In (iii), the SigmaA-weighted 2Fo-Fc map of the final refinement step from autoBUSTER, calculated to 2.3 Å resolution and contoured at 1.1 σ is shown. (c) rCD45d3d4 maps. (i) Ta6Br12-SAD phased, phase-extended and density modified map from SHARP (calculated to 2.45 Å and contoured at 1.1 σ). The two rCD45d3d4 molecules in the crystallographic asymmetric unit are colored yellow and magenta, respectively. (ii–iii) Close-up view of electron density for the residual GlcNAc at residue N502. In (ii), a region of the experimental map in (i) is shown. In (iii), the SigmaA-weighted 2Fo-Fc map of the final refinement from autoBUSTER, calculated to 2.45 Å resolution contoured at 1.1 σ is shown. (d) CD45d1–d4 maps. (i–ii) Stick representation of the final CD45d1–d4 model showing the two molecules in the asymmetric unit. (i) SigmaA-weighted 2Fo-Fc map of the final refinement step from autoBUSTER contoured at 1.0 σ. The model is in atomic colouring (oxygen: red; nitrogen: blue; sulphur: orange; carbon: yellow/red). In (ii) the two CD45d1–d4 molecules per asymmetric unit are colour-coded according to temperature factors (green, low B and red, high B).
Supplementary Figure 2 Glycosylation and conservation at the ‘top’ and base of the modular region of CD45.
(a) Thirteen animal CD45 sequences were aligned using MULTALIN (data not shown). Glycosylation sequons in these sequences were identified using NetNGlyc, and the putatively glycosylated Asn-equivalent positions mapped onto the human CD45d1–d4 crystal structure using the alignment. Sites were colored according to conservation level using the alignment. (b) Electrostatic potential calculated using PyMol (-5 KbT/ec (red) to +5 KbT/ec (blue)) is shown. This analysis identifies a surface with asymmetric charge and N-glycan distribution perhaps used to access substrates (shaded region in c). Views of the d1–d4 region along its long axis from the top (d) and bottom (e) depict: (left) residue conservation at the top and base (residues are colored according to the number of identical or conservatively substituted residues at each position in an alignment of 11 mammalian sequences (data not shown; see Online Methods for residue groupings)); (middle) the network of conserved residues forming the caps (purple, shown in stick format); and (right) the surface electrostatic potential calculated using PyMol (-5 KbT/ec (red) to +5 KbT/ec (blue)). For the sake of clarity, the middle panels in d,e are drawn slightly enlarged relative to the other panels.
(a) Examples of Sema6A-CD45R0 negative-staining EM raw images corresponding to each of the 50 classes shown in (b). Each class contained between 127 and 399 such images. (b) Sema6A-CD45R0 negative-staining EM class averages. The gallery of all fifty class-sums derived from the Sema6A-CD45R0 images (top) and their corresponding I-images (bottom). (c) Fourier Shell Correlation of the reconstruction of Sema6A-CD45R0 made using SPIDER software, showing a resolution at FSC = 0.5 of 33 Å. (d) The original class averages of the Sema6A-CD45R0 negative stain images, prior to 3D reconstruction, compared to reprojections of the reconstruction itself. In each case the class averages are grouped by reference to the reprojections they agree with best. For each block of images (1–24), the final image (marked by yellow borders) is the reprojection best matching the other image(s) shown in that block. (e) Two views (as in Figure 3c of the main text) of the reconstruction fitted with the Sema6A and CD45d1–d4 region and with the mucin linker modeled in between them. Pairs of images are shown, in which the first is the same as in Figure 3c and the second has superimposed a map calculated for the Sema6A-CD45R0 atomic model filtered to 33 Å to show that the mucin linker is unresolved.
Supplementary Figure 4 Distribution of non-chimeric and chimeric forms of Lck versus CD45 and signaling at close contacts.
(a) Additional examples of close contact formation and CD45 segregation observed for J.CaM1.6 T cells expressing mLck. (b) Mean time taken between initial cell contact with OKT3-coated glass surface and calcium release (maximum Fluo-4 fluorescence signal; lag triggering time), at 20°C. (c–f) Additional examples of close contact formation and CD45 segregation observed for J.CaM1.6 T cells expressing TMLck (c), RABCLck (d), R0Lck (e) and d1–d4Lck (f) interacting with an OKT3-coated surface. The Lck constructs were labeled via a HaloTag and HaloTag TMR (red) and CD45 was labeled using Alexa Fluor 488-tagged Gap 8.3 Fab fragments (green). Cell positions were estimated from white-light transmission images (white circles). Imaging data in (a) and (c–f) was taken at 20°C; scale bars, 5 μm. (g) Fractions of J.CaM1.6 T cells expressing the constructs shown in Figure 5a,b of the main text eliciting a calcium response upon contacting OKT3-coated glass at 37°C. Short-, but not the long-forms of Lck rescue OKT3-triggered signaling in J.CaM1.6 T cells. The shaded part of the graph is the 95% confidence interval for the J.CaM1.6 dataset defining the range of values for individual calcium responses indistinguishable from those of the J.CaM1.6 cells. (h) Calcium responses of T cells contacting glass surfaces in the presence and absence of anti-TCR antibody. Glass surfaces were coated with OKT3 antibody or bovine IgG (“IgG”). The fractions of native CD4+ (“CD4+”) and normal or mutant Jurkat T cells expressing or not expressing the TCR that elicited a calcium response upon contacting the coated surfaces at 37°C are shown. The shaded part of the graph is the 95% confidence interval for the J.RT3-T3.5 dataset defining the range of values for individual calcium responses indistinguishable from those of the J.RT3-T3.5 cells. (i) Individual calcium (Fluo-4) intensity profiles for triggered cells contacting OKT3 or IgG-coated glass surfaces at 37°C. (j) Mean intensity profiles of 100 randomly chosen cells triggered in response to contacting OKT3 or IgG-coated glass surfaces at 37°C (mean ± S.D.). The data were collected in at least three independent experiments. Source data
(a) Pixel intensities from images that are anti-correlated when plotted against each other display a poor linear fit, whereas correlated images (b) display a good fit.
(a) Montage from a time-lapse showing the spatio-temporal organization of CD45 (labeled with Alexa Fluor 488-tagged Gap 8.3 Fab, green) during contact of a Jurkat T cell expressing a transmembrane-anchored, non-signaling, T92A-mutated form of CD48 (CD48+) and a SLB containing rCD2 (Alexa Fluor 647-tagged, red). (b) Equivalent to (a) except that the fluorophores are swapped (CD45 is labeled with Alexa Fluor 647-tagged Gap 8.3 Fab, green, and rCD2 is tagged with Alexa Fluor 488, red). (c–e) Montages show distribution of a truncated form of CD45 (HA-CD45, c), TCR (d) and mLck (e) each labeled with HaloTag TMR, red) versus CD45 (green) during contact of a CD48+ Jurkat T cell with a SLB containing rCD2. The time intervals between frames shown are 5 s; data was collected at 37°C. (f) More examples of mLck versus CD45 distribution at T cell contacts with SLBs. Examples of rarer multi-focal contacts of CD48+ Jurkat T cells with rCD2-containing SLBs showing the organization of CD45 (labeled with Alexa Fluor 488-tagged Gap 8.3 Fab, green) and mLck labeled with HaloTag TMR (red).
(a) Zap70 cluster trajectories (colour-coded with time). (b) Schematic depiction of the trajectory analysis: the vector from the center of the contact to the end of a given trajectory is calculated as well as the vector from the start to the end of the trajectory. The angle between the two vectors is 0/180° for centripetal motion, and takes a random value for random motion. (c) Histogram showing the frequencies for all calculated angles γ for Zap70 trajectories observed in Jurkat and JRT3-T3.5 cells during the first 180 s post contact formation with a rCD2 bilayer. Total number of trajectories analyzed: 2234 (Jurkat), 2158 (JRT3-T3.5), total number of analyzed cells: 28 (Jurkat), 36 (JRT3-T3.5). The data were collected in at least three independent experiments.
Supplementary Figures 1–7 and Supplementary Tables 1 and 2 (PDF 7193 kb)
Representative movie (raw data) showing the influx of calcium (change in Fluo-4 intensity) in cells expressing mLck upon interaction with an ‘activating’ surface (OKT3-coated glass) at 20°C. Movie is part of the data presented in Figure 5g. The movie is recorded as 300 ms frames every 1 second and played at 33 frames per second. Scale bar, 10 μm. (AVI 10668 kb)
Representative movie (raw data) showing the influx of calcium (change in Fluo-4 intensity) in cells expressing RABCLck upon interaction with an ‘activating’ surface (OKT3-coated glass) at 20°C. Movie is part of the data presented in Figure 5g. The movie is recorded as 300 ms frames every 1 second and played at 33 frames per second. Scale bar, 10 μm. (AVI 10769 kb)
Representative movie (raw data) showing that mLck and CD45 segregate immediately upon contact of Jurkat T cells expressing CD48 with a rCD2-containing SLB. CD45 is labeled with Alexa Fluor 488-tagged Fab (green) and mLck is labeled with TMR via a HaloTag® (red). The movie is played back 10-fold faster than real-time (10 frames per second). Scale bar, 2 μm. (AVI 10420 kb)
Representative movie (raw data) showing that CD45 exclusion on rCD2-containing SLBs is contact dependent. Normal Jurkat T cells (i.e. cells not expressing CD48) do not form a stable cell-SLB contact: rCD2 in the SLB (left panel) does not accumulate and CD45 expressed by the cells (right panel) is evenly distributed (i.e. there is no exclusion). CD45 is labeled with Alexa Fluor 488-tagged Fab (green) and rCD2 is tagged with Alexa Fluor 647 (red). At 0.18 a white light transmission image of the same area is shown indicating the presence of multiple Jurkat T cells above the SLB. The movie is played back 10-fold faster than real-time (10 frames per second). Scale bar, 2 μm. (AVI 9961 kb)
Representative movie showing simultaneous CD45 exclusion and Zap70 cluster formation in a CD48-expressing Jurkat T cell interacting with a rCD2-containing SLB. The movie combines raw data for the CD45 channel (i.e. CD45 labeled with Alexa Fluor 488-tagged Fab (green), left) with a movie of the Zap70 channel (i.e. Zap70 labeled with HaloTag® TMR, red) prior to (raw data, middle) and post application of a bandpass filter (right; Zap70 clusters identified by the analysis software are marked with a circle and running number). The movie is played back 5-fold faster than real-time (10 frames per second). Scale bar, 2 μm. (AVI 2244 kb)
Movie collage analogous to Supplementary Video 5 for a CD48-expressing J.RT3-T3.5 T cell. The movie is played back 5-fold faster than real-time (10 frames per second). Scale bar, 2 μm. (AVI 2665 kb)
The animation shows the resting T cell surface and the changes in organization of signaling proteins that occur when the cell interacts with an SLB containing the cell adhesion molecule CD2, leading to ligand-independent TCR triggering. The early stages of the animation strive to portray the rapid movements of the proteins and the crowded nature of the T cell surface. The interaction of CD2 (in the SLB) with a non-signaling form of CD48 (on the T cell) results in close-contact formation and the exclusion of CD45. Because contact with the SLB is over a large area, the exclusion of CD45 results in strong TCR phosphorylation by non-excluded Lck kinase, leading to ZAP70 recruitment and calcium signaling in the absence of TCR ligands. The animation finishes with an image of an SLB-contacting Jurkat T cell from the present study showing the distribution of CD45 (green) and Lck (red; see Supplementary Fig. 6f). The molecules shown are: the TCR (terracotta; based on a model using TCRαβ PDB accession code 1AO7, CD3ɛδ PDB 1XIW, CD3ɛγ PDB 1JBJ and CD3ξξ PDB 2HAC), CD45 (light green; based on a model using the CD45 ECD structure described herein and the CD45 phosphatase domain PDB 1YGR); Lck in an active conformation (red; based on Src PDB 1Y57 and the zinc-clasp motif in PDB 1Q68); CD48 (dark green) and CD2 (yellow) modeled on human CD2 (using PDB 1HNF); CD48/CD2 complexes based on a model of the CD2/CD58 complex (using PDB 1QA9); and ZAP-70 (purple; PDB 4K2R). All transmembrane regions and CD3 cytoplasmic regions were modeled as α-helixes in the Build Structure function in UCSF Chimera (http://www.cgl.ucsf.edu/chimera/), using the native protein sequences. A probe radius of 8 Å was used to generate all model surfaces. The animation was built using Blender software (http://www.blender.org). (MP4 131031 kb)
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Chang, V., Fernandes, R., Ganzinger, K. et al. Initiation of T cell signaling by CD45 segregation at 'close contacts'. Nat Immunol 17, 574–582 (2016). https://doi.org/10.1038/ni.3392
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