K118I/K120P CD28 rapidly adopts a conformation that would allow bivalent binding in molecular dynamic simulations.

(A) Structural model of the extracellular domains of the CD28 homodimer illustrating the location of the ligand binding sites (red) and K118/K120 (yellow) at the base of the dimer interface. The K118/K120 residues are only visible on the left hand side monomeric unit in this view. (B) The structural model of CD28 (red) is superimposed on that of CTLA-4 (blue). The illustration shows that the monomeric units are structurally similar, but when forming the dimer, their relative orientations are different. Additional residues including the interchain di-sulfide bond were added at the end of the CD28 structural model shown (see methods for details). (C and D) Three independent trajectories were simulated of WT CD28 (C) or K118I/K120P CD28 (D) with the crystal structure of CD28 as the initial conformation and the RMSD with respect to the initial conformation are shown over time. For simulations of K118I/K120P CD28 (D), there is considerable rearrangement of the subunits at the beginning of the simulations, reflected by an increase in the RMSD. For trajectories 2 and 3, RMSD values start to stabilize towards the end of the simulation. (E and F) CD80 molecules were docked onto the simulated WT CD28 dimers (E) and K118I/K120P CD28 dimers (F), to obtain the corresponding CD28+ CD80 complexes. The surface area buried between carboxy-terminal domains of the docked ligands was calculated at various times. A value of zero indicates no buried surface and thus no contact between the ligands, which would allow for bivalent ligand binding. The fraction of bivalent-competent conformations along each of the three independent trajectories is indicated and the average over all three trajectories was 26% for WT CD28 and 81% for K118I/K120P CD28. (G) A representative conformation from simulations of WT CD28 (red) showing docked CD80 ligands (cyan). It can be seen that the orientation of the ligand binding sites precludes bivalent binding due to steric interference at the distal end of CD80. (H) A representative conformation from simulations of K118I/K120P CD28 (purple) showing bivalently-bound CD80 ligands (cyan).