V600E biases the BRAF kinase domain toward an activation-compatible conformational ensemble through long-range dynamic rewiring.

How oncogenic mutations reshape kinase conformational ensembles to favor aberrant signaling remains incompletely understood. Here, we combined microsecond-scale molecular dynamics simulations, Markov state models, and neural relational inference to investigate how the V600E mutation remodels the dynamics of the inactive-like, nucleotide-bound BRAF kinase domain. V600E did not produce a single fully active conformation but instead biased the kinase toward an activation-compatible conformational ensemble. MSM analysis revealed pronounced enrichment of a dominant metastable state, S3, characterized by a shortened Lys483-Glu501 Cα-Cα distance and a more inward αC-helix arrangement. This mutation-enriched state was kinetically stabilized, as indicated by prolonged mean first passage times for transitions from S3 to the minor states. At the dynamic-network level, V600E reorganized long-range coordination among the P-loop, αC-helix, activation loop, and distal flexible regions and altered preferred model-inferred communication routes connecting these regulatory elements. Together, these results support a model in which V600E preorganizes the inactive-like BRAF kinase domain toward activation-compatible conformations through conformational reweighting and long-range dynamic rewiring. Such conformational preorganization may facilitate oncogenic signaling in the presence of the additional regulatory interactions required for complete kinase activation.