“DFG-Flip” in the Insulin Receptor Kinase Is Facilitated by a Helical Intermediate State of the Activation Loop  Harish Vashisth, Luca Maragliano, Cameron F. Abrams  Biophysical Journal  Volume 102, Issue 8, Pages 1979-1987 (April 2012) DOI: 10.1016/j.bpj.2012.03.031 Copyright © 2012 Biophysical Society Terms and Conditions

Figure 1 (Color online) (a) Key structural features are highlighted in the inactive (1IRK.pdb) and active (1IR3.pdb) crystal structures of the insulin receptor kinase domains. (Transparent cartoons) Protein backbone; (red) A-loop and αC-helix; (cyan and blue) side chains of Asp1150 and Phe1151 of the DFG-motif, respectively. The side chains of residues in R-spine (orange sticks) and C-spine (green sticks), along with their transparent surface-renderings, respectively. (b) Schematic representation of the simulation domain (73 Å × 76 Å × 76 Å) as viewed along the z axis: (blue cartoon) N-lobe and (cyan cartoon) C-lobe; (red) crystallographic water molecules; (yellow spheres) ions; and (wireframe) solvent. The system contains 40,044 atoms in all. Biophysical Journal 2012 102, 1979-1987DOI: (10.1016/j.bpj.2012.03.031) Copyright © 2012 Biophysical Society Terms and Conditions

Figure 2 (Color online) (a) RMSD versus simulation time (ns) for the activation-loop (the A-loop), R-spine, C-spine, and Phe1151 with respect to the active crystal structure for TAMD-run1 (see Table 1). (Gray background in the plots) The first ∼7-ns of MD equilibration, which is followed by ∼40 ns of TAMD. (b) Representative snapshots of IRKD from TAMD simulation are shown at various time-points with the A-loop (red) and side chains of Asp1150 and Phe1151 (cyan and blue), respectively. (Black cartoon) Conformation of the A-loop in the active crystal structure. See Fig. 1 b for the labels and coloring scheme of R- and C-spine residues. (Large panel, center) Conformations of IRKD with highlighted structural motifs: αC-helix, nucleotide-binding loop, and the activation loop from TAMD simulation at t = 7.59 (red), 17.09, 22.89, 40.09, and 47.00 ns (blue). (Arrow) Directional guide along the increasing simulation time. Biophysical Journal 2012 102, 1979-1987DOI: (10.1016/j.bpj.2012.03.031) Copyright © 2012 Biophysical Society Terms and Conditions

Figure 3 (Color online) (a) Interatomic distance-traces for various salt-bridging interactions formed by Lys1030 and Arg1155. (Gray background) The first ∼7 ns of MD equilibration, which is followed by ∼40 ns of TAMD. (Horizontal lines) Distances in the active crystal structure. (b) Snapshots of IRKD highlighting the side chains of residues involved in salt-bridging interactions with the A-loop (shown in red). (c) Snapshots of IRKD highlighting the sequence of solvent-exposure for three the A-loop tyrosines (Tyr1158,1162,1163). (Black cartoon) Conformation of the A-loop in the active crystal structure; (last two panels) the three tyrosine residues. Biophysical Journal 2012 102, 1979-1987DOI: (10.1016/j.bpj.2012.03.031) Copyright © 2012 Biophysical Society Terms and Conditions

Figure 4 (Color online) (a) Free energy profiles along the initial (red) and final converged (black) activation pathways. (Profiles for all string method iterations appear in Fig. S1 in the Supporting Material.) (b) Representative conformations of IRKD from MFEP images 1, 4, 6, 12, 13, 14, 18, and 23, with the A-loop (red), and the side chains of Asp1150 and Phe1151 (cyan and blue, respectively). Biophysical Journal 2012 102, 1979-1987DOI: (10.1016/j.bpj.2012.03.031) Copyright © 2012 Biophysical Society Terms and Conditions