(A) Ribbon representations are shown of the structure of pMHC125 (viewed from the perspective of a TCR). Peptide residues that are available for TCR contact are shown in red, peptide residues occupying shallow surface pockets are shown in green, and peptide residues that serve as MHC anchors are shown in blue. (B) Structural comparison of MBPAc1-11 (gray) and MBP125-135 (green) when bound to I-A^u. (C) Computationally docked model of 1.D9.B2/pMHC125 complex using RosettaDock. MHC α and β chains are depicted in dark blue and light blue respectively with peptide residues in green. TCR chains are shown in yellow (α) and orange (β).

(A) Parental and mutant 1.D9.B2 hybridomas were tested against APCs pulsed with whole MBP, MBP121-140 or MBP131-150 as indicated. Responses are shown as a ratio of mutant to wild-type (parent) responses. Mutations that reduced responses to less than 15% of the parental response (threshold indicated by the dotted line) are considered to have abrogated the response and are therefore defined as hotspots. P-scores for two selected pairs of responses are also shown. (B) The sequence alignment of the variable domains of 172.10 and 1.D9.B2 is shown, with CDR regions indicated and sequence differences boxed. Sequence differences outside CDR3 regions are mutations engineered to increase sc172.10 solubility.

(A) CDR loops for both 172.10 and 1.D9.B2 are shown on I-A^u (CDR1: blue; CDR2: red; CDR3: green; 1.D9.B2 in darker shades and 172.10 in lighter shades). Despite highly conserved loop structures, rotation of the α chain and opening of a groove precludes similar docking on the conserved MHC residues. Computational modeling of 1.D9.B2 with pMHC125 predicts a ∼14° rotation in order to accommodate both structural (αβ interface) and peptide differences (MBP125-135 v. MBPAc1-11). (B) A contact map derived from the modeled 1.D9.B2:pMHC125 complex highlights TCR residues (shown in orange and yellow circles) making specific interactions to pMHC.

(A) ΔΔG and footprint analyses of the interfaces of all six TCR/MHC II complexes currently available are mapped onto molecular surfaces of the TCR and pMHC from each complex, colored by domain (TCRα: light yellow; TCRβ: light orange; MHCα: dark blue; MHCβ: light blue; and peptide: green). Each complex has been splayed open to reveal interface features, with each partner in a complex viewed from the perspective of the cognate partner. For each complex pair, the TCR is shown above the MHC. Hotspot symbols (circles for TCR residues, squares for pMHC residues) are scaled to the magnitude of the contribution (ranging from 1 kcal/mol to 7 kcal/mol), labeled by residue ID and colored according to chain identity. For each surface, the footprint of the binding partner is also outlined as an irregular blob, colored by the molecule contributing the contact: TCRα in yellow, TCRβ in orange, MHCα in dark blue, MHCβ in light blue and the peptide in green. (B) Overlay, based on MHC α-chain superpositions, of the minimum bounding rectangles for TCR footprints on pMHCs. Bounding boxes of TCR footprints from non-autoimmune contexts are shown in orange and autoimmune contexts in green. The center of each box is indicated by a cross. (C) 1.D9.B2 experimental hotspots (Figure 1) are mapped onto the sc1.D9.B2 structure for comparison to the data above (molecular surface colored as in (A); residues where mutations abolish recognition of both ligands are shown as hotspot symbols colored as in (A)). The structure of the disordered CDR3β loop and the footprint of pMHC125 are based on the computationally-docked model (Figure 6). Residues that are distal from the recognition surface are labeled in italics and residues on the recognition surface are labeled in boldface.

(A) Cα backbone representations of the structures of scD10 (Vα2/Vβ8.2; specific for I-A^k/CA), sc172.10 (Vα2.3/Vβ8.2) and all four independent views of sc1.D9.B2 (Vα2.3/Vβ8.2) are shown superimposed on their Vβ domains (in shades of gray; Vα domains are colored as indicated) to highlight the effect of α/β interdomain angle and rotational axis variation. (B) TCR variable domain cassette pseudodyad axes are plotted to graphically highlight distinctions, labeled by TCR name and complex state (U: unbound or B: bound); individual TCRs are grouped by color, with abbreviated labels in a single, matching color if more than one structure is available. For cases where multiple ligand complex structures are available, TCRs are labeled as X1, X2, etc., as listed in Supplemental Table 1. Variation in the pitch of the pseudodyad axes ((ω, φ); Supplemental Table 1) is shown by plotting the intersection of each TCR's associated axis with an arbitrary plane, roughly parallel to the pMHC binding surface (the location of the axes emanate from a single point at the center of mass of the D10 TCR). As a reference, a schematic outline of an idealized TCR is shown (Vα domain on the left (yellow) and Vβ domain on the right (orange)). (C) Variation in the rotation around pseudodyad axes (χ) is plotted, with TCRs labeled as in (B); χ values have been multiplied by five to highlight distinctions. The variation in χ between sc1.D9.B2 (red), 172.10 (orange), and D10 (yellow) is highlighted by coloring the labels; extreme variation in other TCRs is highlighted by boxing labels and coloring lines (1G4 in cyan, ELS4 in dark blue, KB5C20 in green and BM3.3 in light blue).

(A) Ribbon representations of the structures of molecules AB and CD (shown in shades of green or blue, respectively), which together form the diabody present in the AU, are shown; secondary structure elements are shown as arrows (β-strands) and coils (α-helices). Ordered sections of the introduced linker peptides, corresponding to thirteen of fifteen residues total for AD and all fifteen residues for CB, are shown in red; disordered sections of the structure are shown as dashed red lines. (B) Ribbon representations of the structures of molecules EF and GH, which crystallized as monobodies, are shown in shades of orange and red, respectively. Positions of CDR loops are additionally highlighted with CDR1 loops are in blue, CDR2 loops in grey, and CDR3 loops in green. The two molecules have been superimposed to emphasize structural conservation. (C) Superposition of all four molecules in the asymmetric unit highlights a small difference in the α/β interdomain angle between the diabody (in shades of green) and monobody (shades of orange) forms of TCR 1.D9.B2 in the AU, with the diabody molecules displaying a slightly more extreme α/β interface angle. (D) The conservation of CDR structure is shown by all-atom representations of superpositions of the four views of each 1.D9.B2 CDR along with the corresponding element from 172.10 where applicable. 1.D9.B2 CDRs are colored to indicate results of the scanning mutagenesis (wild-type level response by mutant: blue; ablation of response: red; and not tested: black); 172.10 CDRs are colored by atom type (carbon: light gray; nitrogen: blue; oxygen: red). For CDR1α, CDR2α, and CDR1β, TCR 172.10 (shown in white) adopted the same conformation. (E) Four CDR3 amino acid changes between 1.D9.B2 and 172.10 result in the loss or gain of interdomain contacts. Residues labeled in red (α93, α103, α104) represent loss of contacts along the groove face whereas the last panel, labeled in green, highlights a gain of contact (β107/110) on the face opposite the groove. In the panels, 1.D9.B2 is depicted in yellow (α chain) and orange (β chain). Light green (α chain) and dark green (β chain) residues are from 172.10.