WT CD28 adopts a bivalent binding conformation when starting from the CTLA-4 dimer orientation.

MD simulations were run with WT CD28 starting from a CTLA-4 dimer orientation. (A) The RMSD of three independent trajectories with respect to the initial conformation are shown over time. There is considerable rearrangement of the subunits at the beginning of the simulations, reflected by an increase in the RMSD. The conformational fluctuations stabilize after the first 100 ns of simulation, although the trajectories adopt different conformations. This instability was not inherent to this dimer orientation, as the RMSD of MD simulations of CTLA-4 remained below 4Å (data not shown). (B) CD80 molecules were docked onto the simulated CD28 dimers to obtain the corresponding CD28-CD80 complexes. The surface area buried between the docked ligands was calculated at various times along each of the three independent trajectories to estimate the fraction of bivalent-competent conformations; the average over all three trajectories was 79%. (C) A representative conformation of CD28 (green) showing docked CD80 ligands (cyan) illustrates the potential for bivalent ligand binding.