Fig. 6: The flip in the neoantigen occurs via an under-peptide motional pathway resembling a limbo dance.

a Energy diagram describing the peptide conformational change as a transition from a lower to higher energy flipped state, resulting in a high energy barrier in the forward direction and a low barrier in the reverse direction. b Under-peptide motional pathway illuminated by the reverse WEMD simulations beginning with the conformation in the ternary complex with TCR4, showing pTrp6 of the neoantigen moving underneath the peptide backbone. The three under-peptide conformations are extracted from three roughly equally spaced time points of a single successful transition. c Solvent accessible surface area of the pTrp6 side chain from each frame of the 109 successful reverse WEMD transitions. The solid cyan-to-yellow curve was generated from LOWESS smoothing of the data. The value expected from an exposed side chain if pTrp6 transitioned by moving over the top of the peptide (~120 Ų) is indicated by the arrow. d RMSD of pTrp6 from the TCR4-bound conformation in SMD simulations of the peptide/HLA-A3 complexes forcing under- or over-peptide rotations. Data are shown for both the neoantigen and the WT peptide as a function of progressively larger spring constants. Only the neoantigen in an under-peptide rotation reaches the bound state, as indicated by the top row. e Number of unfavored ϕ/ψ torsion angles for all non-terminal amino acids of the neoantigen in the under- or over-peptide SMD simulations. Attempting to force an over-peptide rotation by increasing the spring constant results in greater torsional resistance. f Distance between the center of mass (COM) of the pTrp6 side chain and the position 2 side chain in the WT peptide (left) or neoantigen (right) under-peptide SMD simulations, colored by degree of van der Waals (vdW) overlap between the side chains. In the neoantigen simulation, the side chains remain distant, with little to no atomic overlap. In the WT peptide simulation, the side chains come in close proximity, with substantial overlap as the simulation progresses. Data are from the simulations with the 100 kJ/mol/mm² spring constant. Bracketed values give the average atomic overlap in Å.