STAT3 structure.

1BG1 was used as the template; NTD is not shown. (A) STAT3 domain structure (Y705 is shown as spheres, D170 is shown as sticks). Vide infra for details of the initial structure.(B) Secondary structures are labeled according to the UniProt database (S1 Table) [6]: α helices are colored blue, β sheets are colored red, and unstructured regions (loops) are colored yellow (transverse view). The assigned secondary structures combine information from multiple x-ray crystal structures, thus there is some mismatch with the specific structures used in this work.</p

CCD <i>α</i>3 global tilt angle distribution.

(A,B) CCD α3 global tilt angle distribution of different macrostates, plotted separately for the wild type and D170 variant. Average helix tilt angle within each macro-state is illustrated by a vertical dashed line. (C,D) CCD α3 global tilt angle distribution of different replicas for wild type and D170A variant. (TIF)</p

The PCA analysis of pair distances of rigid core without residue 562 (residues:240–252, 474, 479, 511, 546, 549, 550, 564, 568, 610, 611).

(A) PCA 2D plot colored by different macrostates; (B) PCA 2D plot colored by systems; (C) The first ten features that contribute the first two PC the most. The absolute value of PC1 and PC2, and square root of sum of squared PC1 and PC2 values are shown here. (TIF)</p

Rigid core analysis showing interactions which are conserved across all macro-states.

The analysis includes all trajectories spanning macro-states M0–M4. (A) Distance distributions of the atom pair, that form hydrogen bonds, show similar distribution profiles. (B)Right: Inter-domain pair Cα distances (dashed lines) shows the intensity of coupling in the Cα pair motions between different domains. The pair Cα distances are colored from blue, green, yellow, and orange based on their deviation throughout the trajectory (Map in subplot). Left: A close-up of multi-domain interaction site shows the least first 200 deviating Cα pairs, demonstrating strongly correlated movements among CCD, LD and SH2 domain. (C) Conserved hydrogen bond network between CCD, LD, and DBD. (D) Conserved π-π interaction network between LD and DBD. (E) Conserved hydrogen bond network as well as hydrophobic interactions between LD and SH2. (F) Distribution of global helical tilt of α3 in CCD for each macro-state (M0–M4, see also supplemental S6 Fig). Statistical tests (T-test and Kolmogorov-Smirnov test) are shown in Table A in S1 Text</p

Additional information of PCA and MSM analysis.

(A) PCA scree plot: dot shows the cumulative explained variance of the principal components; the bar chart represents the explained values per component. (B) Relaxation timescales of MSM for SH2 domain conformational space at different lag times. (C) The first ten features that contribute the first two PC the most. The absolute value of PC1 and PC2, and square root of sum of squared PC1 and PC2 values are shown here. (TIF)</p

Additional REDAN analysis results.

(A) Proposed pathway from 170 to 640 shown in the protein structure; (B) Proposed pathway from 170 to 644; (C,D,E) Key pair residue distance; (F) Ramachandran Dihedral for residue 513,514, 515 and 517; (G) Summary of proposed pathways from source residue 170 to target residue 640,644 and 657. (TIF)</p

The mean RMSF of six replicates was plotted, with the RMSF values for each residue separated by domain.

The wild type is plotted in blue and the D170A variant in orange, with error bar indicating the stand error among the six replicates. Structures of each domain colored by wild type RMSF values are shown (low RMSF values in white to high RMSF values in red). (TIF)</p

Figure depicting the simulation system, generated by VMD.

Water is shown as lines, ions are shown as vdw, protein is shown as cartoon. (TIF)</p

System parameters (pressure, temperature) and system energy (potential energy, kinetic energy and total energy) of wild system and D170A variant system, each system has six replicas: cp1, cp2, cp3, cp4, cp5 and cp6.

(TIF)</p

A close-up view of the hydrogen bond network changes between macro-states M0–M3 and macro-state M4 (see Fig 5 for details).

(A—C) Differential rate of pair residue hydrogen bonds in the LD and SH2 domains. (D—F) Differential rate of pair residue hydrogen bonds in the CCD and LD domains.</p