Identification of the Docking Site for CD3 on the T Cell Receptor β Chain by Solution NMR*

Background: Understanding T cell signaling requires knowing the structure of the TCR-CD3 complex. Results: Solution NMR was used to identify the docking site for CD3 ectodomains on the TCR β chain. Conclusion: The docking site (∼400 Å2) comprises ∼10 Cβ residues at the base of the TCR. Significance: CD3 is located opposite to the peptide-MHC binding site of the TCR. The T cell receptor (TCR)-CD3 complex is composed of a genetically diverse αβ TCR heterodimer associated noncovalently with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates peptide-MHC recognition, whereas the CD3 molecules transduce activation signals to the T cell. Although much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by peptide-MHC initiates signaling is poorly understood. A key to solving this problem is defining the spatial organization of the TCR-CD3 complex and the interactions between its subunits. We have applied solution NMR methods to identify the docking site for CD3 on the β chain of a human autoimmune TCR. We demonstrate a low affinity but highly specific interaction between the extracellular domains of CD3 and the TCR constant β (Cβ) domain that requires both CD3ϵγ and CD3ϵδ subunits. The mainly hydrophilic docking site, comprising 9–11 solvent-accessible Cβ residues, is relatively small (∼400 Å2), consistent with the weak interaction between TCR and CD3 extracellular domains, and devoid of glycosylation sites. The docking site is centered on the αA and αB helices of Cβ, which are located at the base of the TCR. This positions CD3ϵγ and CD3ϵδ between the TCR and the T cell membrane, permitting us to distinguish among several possible models of TCR-CD3 association. We further correlate structural results from NMR with mutational data on TCR-CD3 interactions from cell-based assays.

The T cell receptor (TCR)-CD3 complex is composed of a genetically diverse ␣␤ TCR heterodimer associated noncovalently with the invariant CD3 dimers CD3⑀␥, CD3⑀␦, and CD3. The TCR mediates peptide-MHC recognition, whereas the CD3 molecules transduce activation signals to the T cell. Although much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by peptide-MHC initiates signaling is poorly understood. A key to solving this problem is defining the spatial organization of the TCR-CD3 complex and the interactions between its subunits. We have applied solution NMR methods to identify the docking site for CD3 on the ␤ chain of a human autoimmune TCR. We demonstrate a low affinity but highly specific interaction between the extracellular domains of CD3 and the TCR constant ␤ (C␤) domain that requires both CD3⑀␥ and CD3⑀␦ subunits. The mainly hydrophilic docking site, comprising 9 -11 solvent-accessible C␤ residues, is relatively small (ϳ400 Å 2 ), consistent with the weak interaction between TCR and CD3 extracellular domains, and devoid of glycosylation sites. The docking site is centered on the ␣A and ␣B helices of C␤, which are located at the base of the TCR. This positions CD3⑀␥ and CD3⑀␦ between the TCR and the T cell membrane, permitting us to distinguish among several possible models of TCR-CD3 association. We further correlate structural results from NMR with mutational data on TCR-CD3 interactions from cell-based assays.
X-ray crystallographic studies of TCR-pMHC complexes have revealed the molecular basis for TCR recognition of foreign antigens and self-antigens (4 -8). In addition, much is known about the downstream signaling cascade, following phosphorylation of CD3 ITAMs by the Src kinase Lck associated with CD4 or CD8 (9). However, the mechanism(s) by which TCR ligation is communicated to the CD3 signaling apparatus remains a mystery (10,11). A critical missing element to solving this puzzle is knowledge of the spatial organization of the TCR-CD3 complex and the precise interactions between TCR and CD3 subunits. Although structures of the immunoglobulin (Ig)-like ectodomains of isolated CD3⑀␥ and CD3⑀␦ heterodimers have been determined (12)(13)(14)(15), exactly how these subunits associate with TCR on the T cell surface remains to be established.
A variety of approaches have been used to infer the stoichiometry, pairing, and orientation of subunits within the TCR-CD3 complex. For example, mutational studies have demonstrated the importance of conserved charged residues in the transmembrane helices of the TCR and CD3 chains for complex assembly (1,16). The following pairings have been established: TCR␣-CD3⑀␦, TCR␣-CD3, and TCR␤-CD3⑀␥. Besides these transmembrane interactions, extracellular interactions between the TCR and the Ig-like ectodomains of CD3⑀␥ and CD3⑀␦ also contribute to the structural integrity and function of the TCR-CD3 complex (2,11,(17)(18)(19)(20)(21). By contrast, the ectodomain of CD3, which is only 9 residues long, has not been implicated in interactions with the TCR. Although possible docking sites on the TCR for CD3 subunits have been proposed (18 -20), these key interaction regions remain undefined.
Recently, small angle x-ray scattering (SAXS) was applied to a soluble TCR-CD3⑀␦ complex that was engineered for stability by replacing the transmembrane domains with a heterotrimeric coiled coil (22). The SAXS results indicated that the extracellular domains (ECDs) of CD3⑀␦ are situated underneath the TCR ␣ chain, although a docking site was not identified. This arrangement was consistent with negative stain electron microscopy (EM) of the entire TCR-CD3 integral membrane complex, which further showed that the CD3⑀␥ ECDs sit beneath the TCR ␤ chain (22). Intriguingly, EM images revealed mainly dimeric complexes in which two TCRs projected outward from a central core composed of the CD3 ECDs.
Although SAXS and negative stain EM have provided important information on the overall organization of the TCR-CD3 complex, these structural techniques lack the resolution necessary to establish the precise orientation of TCR and CD3 subunits in the complex or to define TCR-CD3 interfaces. At present, such information can only come from x-ray crystallography, NMR, or cryo-EM, applied individually or in combination. However, efforts to crystallize TCR-CD3 complexes have been thwarted by the very low affinity of TCR-CD3 interactions in solution. Similarly, previous attempts to map binding epitopes by NMR were unable to detect an interaction between TCR and CD3 ECDs (14). Using optimized protein constructs and isotope labeling conditions, we have revisited the application of NMR to define TCR-CD3 contact sites in solution. Here we demonstrate a specific interaction between TCR and CD3⑀␥ and CD3⑀␦ ECDs by NMR, identify the docking site for CD3 on the TCR ␤ chain, and correlate our structural results with mutational data on TCR␤-CD3 interactions from cell-based assays.

Experimental Procedures
Production and Purification of CD3⑀␥ and CD3⑀␦ Heterodimers-Human CD3⑀␥ and CD3⑀␦ heterodimers were prepared by in vitro folding from inclusion bodies produced in Escherichia coli. CD3⑀␦ needed to be folded together with the anti-CD3⑀ antibody UCHT1 in order to form a stable species (15). By contrast, the CD3⑀␥ heterodimer was stable on its own. DNA fragments encoding the ECDs of CD3⑀ (residues 1-105), CD3␥ (residues 1-90), and CD3␦ (residues 1-79) were inserted separately into the vector pET-26b (Novagen) and expressed as inclusion bodies in BL21(DE3) E. coli cells (Novagen). The UCHT1 antibody was likewise produced in inclusion bodies as a single-chain Fv fragment (scFv) in which the light chain variable region was connected to the heavy chain variable region by a (GGGGS) 3 linker. Bacteria were grown at 37°C in LB medium to A 600 ϭ 0.6 -0.8 and induced with 1 mM isopropyl-␤-D-thiogalactoside. After incubation for 3 h, the bacteria were harvested by centrifugation and resuspended in 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl and 2 mM EDTA; cells were disrupted by sonication. Inclusion bodies were washed extensively with 50 mM Tris-HCl (pH 8.0) and 2% (v/v) Triton X-100. The CD3⑀, CD3␥, and CD3␦ chains were dissolved in 8 M urea, 50 mM Tris-HCl (pH 8.0), and 10 mM DTT; UCHT1 scFv was dissolved in 6 M guanidinium HCl, 100 mM Tris-HCl (pH 8.0), and 10 mM DTT.
Production of TCR MS2-3C8 with Isotope-labeled ␤ Chain-The ␤ chain of TCR MS2-3C8 (residues 1-244) with U-2 H, 13 C, 15 N labeling was obtained by inclusion body expression in E. coli BL21(DE3) cells (Agilent) transformed with pET-26b. Transformed cells were grown in 25 ml of LB medium containing 30% D 2 O (Isotec) at 37°C until A 600 Ն 1.0 and then transferred in a 1:50 dilution to 25 ml of M9 medium containing 70% D 2 O, 1 g/liter [ 15 N]NH 4 Cl (Cambridge Isotope) as the sole source of nitrogen and 4 g/liter [ 2 H, 13 C]glucose (Cambridge Isotope) as the sole source of carbon. The culture was grown until A 600 ϭ 0.5-1.0 and was transferred in a 1:100 dilution to 100 ml of M9 containing 100% D 2 O and grown overnight. The overnight cultures were used to inoculate 1 liter of M9/D 2 O to a starting of A 600 ϭ 0.10. Induction with isopropyl-␤-D-thiogalactoside to a final concentration of 1 mM was performed at A 600 Ն 0.6, and growth was continued for 3-4 h at 37°C. The bacteria were disrupted by sonication. Inclusion bodies were washed with and without 5% (v/v) Triton X-100 and then solubilized in 8 M urea, 50 mM Tris-HCl (pH 8.0), and 10 mM DTT.
For in vitro folding, the TCR MS2-3C8 ␣ and ␤ chains were mixed in a 3.6:1 molar ratio and diluted into a folding mixture containing 5 M urea, 0.4 M L-arginine HCl, 5 mM EDTA, 3.7 cystamine dihydrochloride, and 6.6 mM cysteamine to a final concentration of 50 mg/liter. The folding mixture was dialyzed against H 2 O for 72 h at 4°C and then dialyzed against 10 mM Tris-HCl (pH 8.0) for 48 h at 4°C. After removal from dialysis, the folding mixture was concentrated and dialyzed against 50 mM MES (pH 6.0) at 4°C overnight. Disulfide-linked TCR MS2-3C8 heterodimer was purified using sequential MonoQ and Superdex S-200 columns.
[ 1 H, 12 C]glucose (Sigma) was used as the sole carbon source, and 50 mg/liter of each labeled amino acid (Sigma/Isotec) was added to M9/D2O 1 h prior to induction. A U-2 H, 15 N-labeled MS2-3C8 ␤ chain for titration experiments was produced similarly except for the use of [ 1 H, 12 C]glucose as the sole carbon source.

NMR Assignment of TCR MS2-3C8␣[␤-2 H, 13 C, 15 N]-For
detailed NMR studies, we used a human autoimmune TCR, MS2-3C8, which recognizes a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC class II molecule HLA-DR4 (30). The pathogenic potential of MS2-3C8 was demonstrated using mice transgenic for this TCR and HLA-DR4 (31). An x-ray crystal structure of TCR MS2-3C8 bound to its pMHC ligand, MBP-HLA-DR4, has been determined (30), as well as that of the ternary complex formed by MS2-3C8, MBP-HLA-DR4, and CD4 (32). The interactions of MS2-3C8 with several of the key players in T cell signaling are therefore well characterized structurally, making this TCR a good candidate for studying critical associations with CD3⑀␥ and CD3⑀␦. The MS2-3C8 TCR employed here was modified slightly from the wild-type used for previous x-ray studies, with three mutations in the ␣ chain and four mutations in the ␤ chain. The mutations were made to improve either expression (V␤T55A), folding yield, and stability (C␣T159C, C␣C206(deleted), C␤S171C, C␤S189A, and C␤C245(deleted)) or binding to HLA-DR4 (V␣A31T). None of these mutations are at positions that contact CD3 (see below). Thus, the TCR MS2-3C8 ectodomain studied here is a heterodimer consisting of an unlabeled 205-residue ␣ chain and a 2 H, 13 C, 15 N-labeled 244-residue ␤ chain for a total molecular mass of ϳ50 kDa. Due to this relatively high molecular mass, transverse relaxationoptimized (TROSY)-based triple resonance experiments were utilized for the NMR assignment process (33). Interresidue connectivities were obtained through sequential matching of C ␣ and C ␤ chemical shift patterns and confirmed with CO correlations in most cases. A selectively labeled ILV sample provided further validation of the assignments using methods described previously (34). Overall, 200 (87%) of the 230 possible main chain amide assignments were obtained, with extensive coverage of both TCR variable ␤ (V␤) and constant ␤ (C␤) domains (Fig. 1, A-D). Assignments of H N , 15  TCR MS2-3C8 ␤ Chain Conformation and Dynamics in Solution-Chemical shift index analysis (35) using the assigned C ␣ , C ␤ , and CO resonances identified the secondary structure elements for the MS2-3C8 ␤ chain in solution (Fig. 2A). These results were compared with the x-ray structure of MS2-3C8 in complex with MBP-HLA-DR4 (Protein Data Bank accession code 3O6F) (30). The only region that appears to be slightly different is strand D in the C␤ domain, which is a short 3-residue ␤-strand in the x-ray structure but has a coil conformation in solution. Interestingly, this is the region where we engineered a C␣Cys 159 -C␤Cys 171 interchain disulfide (36) to increase yields of paired TCR ␣␤ heterodimers for the NMR sample. The wild-type protein used for x-ray analysis did not contain this disulfide (30). Generally, however, the secondary structure elements derived from chemical shifts are very similar to those observed in the x-ray structure, indicating comparable fold topologies for MS2-3C8 in solution and crystal forms. The chemical shift data were also used to obtain insights into the flexibility of the ␤ chain in the context of the TCR ␣␤ heterodimer (37). Thus, the high order parameters (S 2 ) for residues in the ␤ chain of MS2-3C8 correspond with more rigid secondary structure elements, whereas lower values reflect less structured and more mobile loop regions (Fig. 2B). Moreover, most high S 2 regions in MS2-3C8 correlate with the lower temperature (B) factors of more ordered regions in the x-ray structure of the MS2-3C8-MBP-HLA-DR4 complex (Fig. 2, B and C). There are two notable exceptions, however. First, the complementarity-determining region loops have low S 2 values, consistent with being disordered in the free MS2-3C8 in solution, but have low B factors, consistent with order in the x-ray structure. This difference between the NMR and x-ray observations is to be expected because complexation with MBP-HLA-DR4 in the x-ray study rigidifies the complementarity-determining region loops, whereas the MS2-3C8 is uncomplexed in the solution NMR study. Second, there is a distinct difference between the main chain mobility inferred from order parameters versus B factors for the two ␣-helical regions in the C␤ domain, ␣A and ␣B (Fig. 2, B and C). For MS2-3C8 alone in solution, the relatively high S 2 values (ϳ0.9) indicate that ␣A and ␣B are ordered ␣-helices. In contrast, these structured helical regions have high B factors (ϳ120 -140) in the x-ray structure of the MS2-3C8-MBP-HLA-DR4 complex, indicating increased mobility relative to other secondary structured regions in the complex. The differences in main chain flexibility of the ␣A and ␣B helices in free and pMHC-bound states are discussed further below.
TCR MS2-3C8 Interactions with CD3 ECDs-Unlabeled samples of recombinant CD3⑀␥ and scFv-CD3⑀␦ were checked for proper folding using one-dimensional 1 H NMR and circular dichroism spectra. 15 N-Labeled samples were also prepared, and two-dimensional 1 H-15 N HSQC spectra indicated well folded proteins (Fig. 3). The addition of unlabeled CD3⑀␥ to MS2-3C8␣[␤-2 H, 15 N] gave minimal chemical shift perturbations, even at the highest TCR/CD3⑀␥ molar ratio of 1:5.8 (Fig.  4, A and B). Most shifts in the NMR spectrum had ⌬␦ total values less than 0.01 ppm, which is within the estimated experimental error of measurement. The few small changes that did occur were mainly due to residues in the C␤ domain, in the ␣A and ␣B helical regions and adjoining loops. Similarly, very few differ-  15 N] (blue) is superimposed on the two-dimensional HSQC spectrum (black). Peaks labeled in blue are due to ILV residues or residues with an (i Ϫ 1) ILV. ences in the MS2-3C8␣[␤-2 H, 15 N] NMR spectrum resulted when only unlabeled scFv-CD3⑀␦ was added. The largest change (⌬␦ total ϳ0.013 ppm) corresponded to Arg 195 in the C␤ domain, a residue that is at the interface with the C␣ domain (Fig. 4, A and B). In contrast to the titrations with separate CD3 ECDs, the binding experiments where CD3⑀␥ and scFv-CD3⑀␦ were added together to MS2-3C8␣[␤-2 H, 15 N] produced more extensive chemical shift perturbations. In this case, 32 residues gave ⌬␦ total values ranging from 0.01 to 0.045 ppm (Fig. 4, A and B) such that 4 were in the V␤ domain and 28 were in the C␤ domain. These changes are consistent with a low affinity but highly specific interaction between CD3 ECDs and the TCR ␤ chain, where only a subset of the NMR peaks are affected upon ligand binding. Moreover, the finding that both CD3⑀␥ and CD⑀␦ must be added together to produce significant chemical shift perturbations suggests that the CD3 ECDs bind cooperatively to the TCR.
Control experiments were also carried out where two-dimensional 1 H-15 N HSQC spectra were acquired at different TCR concentrations ranging from 75 to 250 M. Almost all of the TCR ␤ chain residues have backbone amide chemical shifts that are invariant (⌬␦ total Յ 0.005 ppm) over this range, with a few exceptions in the V␤ domain. The largest ⌬␦ total value in these control experiments was for His 83 (ϳ0.03 ppm) with smaller values (0.005-0.01 ppm) for Met 45 , Asp 86 , Gly 96 , and Gly 97 . The chemical shift perturbations observed for these residues in the CD3 titrations may therefore have some contribution from small changes in TCR concentration-dependent effects.
Mapping of the most perturbed residues onto the x-ray structure of MS2-3C8 showed significant clustering in the C␤ domain, particularly in the ␣A and ␣B helices and neighboring residues, strongly suggesting that this region contains the binding epitope for CD3 (Fig. 5, A and B). Analysis of peak intensities indicated that decreases were uneven as CD3⑀␥ and scFv-CD3⑀␦ were added, with larger losses corresponding to the two ␣-helices in the C␤ domain, ␣A and ␣B (Fig. 4C). Such disproportionate and localized decreases in peak intensity are indicative of binding interactions and provide further support for the chemical shift perturbation data. Based on the intensity changes, a K D was estimated for the interaction between MS2-3C8 and CD3⑀␥/⑀␦ of ϳ320 Ϯ 100 M (Fig. 6). The TCR ␤ chain binding epitope thus identified by NMR is centered on the ␣A and ␣B helices, which are located at the base of the TCR (Fig. 5, A and B). This would position CD3⑀␥ and CD3⑀␦ between the TCR and the T cell membrane, in agreement with the shorter length of the CD3 stalks compared with the TCR ␣ and ␤ chain stalks. The epitope is mainly hydrophilic, including the following solvent-accessible residues: Glu 132 , Ser 136 , His 137 , Ser 197 , Thr 199 , Phe 200 , Asn 203 , Arg 205 , Asn 206 , and possibly also Trp 240 and Arg 242 . The surface area is relatively small (ϳ400 Å 2 ), consistent with the weak interaction between TCR and CD3, although interactions from the ␣ chain are as yet undetermined and may also contribute to binding with CD3. Importantly, the TCR ␤ chain binding epitope is devoid of glycosylation sites, which are located well away from the ␣A/␣B docking site with CD3.
A number of the residues undergoing chemical shift perturbations are not solvent-accessible and so cannot directly contact CD3. Notably, many of these are at the interface between the ␤ and ␣ chains of the MS2-3C8 TCR. Given that the chemical shift is an exquisitely sensitive measure of structure, these changes may reflect small adjustments in orientation between the ␣ and ␤ subunits due to cooperative binding of CD3⑀␥ and CD3⑀␦. Even binding of CD3⑀␥ and scFv-CD3⑀␦ individually to MS2-3C8 both appear to contribute to weak perturbations at the ␣/␤-interface (e.g. Arg 195 for scFv-CD3⑀␦ and Val 125 , Phe 128 , and Ser 131 for CD3⑀␥). If small domain rearrangements between the ␣ and ␤ chains of TCR are important, then it is possible that the non-native interchain disulfide between C␣Cys 159 and C␤Cys 171 may influence CD3 binding. Small long range effects like those seen here are commonly observed in chemical shift mapping of protein binding interactions (38).
Comparison of Binding Epitope with Mutational Data on TCR-CD3 Interactions-The docking site for CD3 on the TCR ␤ chain identified by NMR (Fig. 5, A and B) may be compared with information on this site obtained from cell-based assays. In an extensive analysis of TCR␤-CD3 interactions, Fernandes et al. (20) mutated all C␤ residues whose side chains are Ͼ50% solvent-exposed and tested the effect of these mutations on surface expression of the TCR-CD3 complex in transduced Jurkat T cells lacking a functional TCR ␤ chain. In Fig. 5, C and D, mutations in C␤ that reduced TCR expression by Ͼ60% compared with wild type are shown mapped onto the x-ray structure of MS2-3C8. Strikingly, both the NMR and cell-based approaches identified residues in the ␣A and ␣B helices as interacting with CD3. However, mutations that affected TCR surface expression were broadly distributed over the C␤ domain, in sharp contrast to the clustering of residues showing the largest chemical shift perturbations at the base of C␤ (Fig. 5,  A and B). We conclude that the higher number of C␤ residues implicated by mutagenesis (20) than by NMR indicates that reduced TCR surface expression most likely reflects not only disruption of TCR-CD3 contacts but also deleterious effects on TCR folding, stability, and/or intracellular trafficking.
Impact of Mutations in the TCR␤-CD3 Interface on Cell Surface Expression-Finally, we assessed the importance of the TCR␤-CD3 interaction interface that we defined by NMR on TCR-CD3 cell surface expression. Because the key TCR␤ residues at this interface are conserved between mice and humans (Val 166 /Thr 199 /Asn 203 /Arg 205 ), we made non-conservative mutations in the H-2E k -restricted, moth cytochrome c 88 -102-specific AND TCR at these residues to determine whether this led to the disruption of the TCR␤-CD3 interaction. Multicistronic 2A-peptide-linked vectors were generated that contained the AND TCR␣␤ with either a wild type or mutant TCR␤ (TCR␤ WT or TCR␤ Mut (V166R, T199R, N203R, or R205E)), and an IRES.mAmetrine cassette to facilitate detection of transfectants by flow cytometry (26 -29). Wild type or mutant AND TCR-encoding vectors were co-transfected with a similarly constructed multicistronic vector containing all four wild type CD3 chains (CD3⑀ WT /CD3␦ WT /CD3 WT /CD3␥ WT ) that included an IRES-GFP cassette into HEK 293T cells. Cells were gated on GFP/mAmetrine, and the mean fluorescence of CD3⑀ and TCR␤ was determined. A substantial reduction in TCR-CD3 expression (ϳ66%) was reproducibly observed with TCR␤ Mutversus TCR␤ WT -expressing cells (Fig. 7). These data suggest that this interface is required for optimal TCR-CD3 cell surface expression.

Discussion
We have demonstrated the feasibility of using NMR chemical shift mapping to define contacting surfaces between TCR and CD3 in solution, despite the low affinity of the interaction (K D ϳ320 M). An earlier NMR study did not detect binding between an unlabeled mouse TCR and 15 N-labeled CD3⑀␦ (14). In fact, this result is consistent with our findings that the addition of unlabeled CD3⑀␦ or CD3⑀␥ alone to MS2-3C8␣[␤-2 H, 15 N] produced minimal chemical shift changes and that significant perturbations necessitated the addition of both CD3⑀␦ and CD3⑀␥. This suggests that CD3 ECDs bind cooperatively to the TCR, in agreement with evidence from cell-based mutagenesis experiments that extracellular TCR-CD3⑀␥ interactions require TCR-CD3⑀␦ interactions for stabilization (18). Coopera- tivity could also arise from CD3⑀␥-CD3⑀␦ interactions, although direct contacts between CD3 ECDs remain to be shown.
We have shown that CD3 docks on the ␣A and ␣B helices of C␤. This contact site positions CD3 at the base of the TCR, rather than alongside it, in agreement with results from SAXS and negative stain EM (22). As a consequence, CD3 would be situated between the TCR and T cell membrane, which is consistent with the greater length of the TCR ␣ and ␤ chain stalk regions (21 and 16 residues, respectively) than those of CD3⑀␥ and CD3⑀␦ (each ϳ9 residues). Mutational studies have demonstrated the importance of these highly conserved stalks for the assembly and function of the TCR-CD3 complex (41)(42)(43)(44)(45). Extension of our NMR analysis to the MS2-3C8 ␣ chain will define the docking site for CD3 ECDs on the TCR more completely.
X-ray studies have shown that the ternary complex formed by MS2-3C8, MBP-HLA-DR4, and the CD4 co-receptor resembles a pointed arch in which TCR and CD4 are each tilted relative to the T cell membrane (Fig. 8) (32,46). This structure, in conjunction with the location of the CD3 docking site established by NMR, places CD3⑀␥ and CD3⑀␦ inside the TCR-pMHC-CD4 arch, wedged between the TCR and T cell membrane and facing CD4 (Fig. 8). Importantly, this particular arrangement would achieve intracellular juxtaposition of CD3 ITAMs with the tyrosine kinase Lck bound to CD4, thereby promoting phosphorylation of CD3 ITAMs and T cell triggering.
Although virtually all regions of the MS2-3C8 ␤ chain that were ordered in the NMR structure (high S 2 values) were also ordered in the x-ray structure (low B factors) (30), intriguing exceptions are the ␣A and ␣B helical regions that constitute the CD3 docking site. These ␣-helices are ordered in solution but display high B factors in the crystal. Whereas MS2-3C8 was bound to its pMHC ligand (MBP-HLA-DR4) in the x-ray structure, it was unbound in the NMR study. It is therefore tempting to speculate that binding of pMHC to the TCR could increase   the flexibility of the CD3 docking site, thereby transmitting an allosteric signal from V␤ to C␤ and its associated CD3 subunits. Indeed, recent studies in several systems have demonstrated that ligand binding can alter protein flexibility at distant sites, resulting in long range transmission of biological signals, even in the absence of crystallographically observed structural changes (47)(48)(49)(50). This process, known as dynamic allostery, is of special interest in cases such as the TCR, where x-ray crystallographic studies of multiple TCRs in free form and bound to pMHC have so far failed to identify clear and consistent conformational changes in the TCR C␣ or C␤ domains that could be unambiguously attributed to antigen binding (4,8,10). Future studies will examine the possibility that MBP-HLA-DR4 ligation by MS2-3C8 induces allosteric changes in TCR dynamics that are transmitted to the CD3 signaling apparatus.