Molecular Mechanisms of Gain-of-Function Mutations in λ Cro Revealed by Molecular Dynamics Simulations.

Transcription factors regulate gene expression by coordinating complex networks in organisms ranging from bacteriophages to humans. Bacteriophage λ Cro is a 66-residue repressor that binds DNA as a dimer to block transcription. Because of its small size, simple structure, and well-characterized function, Cro has long served as a model system for understanding the structure/function relationship in transcription factors. Experiments have shown that a small set of mutations can convert it into a dual-function transcription factor capable of both repression and activation. One engineered variant retains activity when truncated to 63 amino acids but loses function at 59, highlighting how little sequence is required for complex regulatory behavior. To probe the molecular basis of this adaptability, we performed multimicrosecond all-atom molecular dynamics simulations of wild-type Cro and two engineered variants, Act3 and Act8. The simulations reveal that minimal sequence changes can reorganize interaction surfaces, shift DNA-binding modes, modulate binding affinities, and redistribute intramolecular communication pathways. These effects on DNA binding occur alongside changes that may broaden regulatory potential, offering insight into how compact transcription factors evolve new functions. Together, these observations provide a mechanistic framework for understanding how transcription factor sequence, structure, and dynamics reshape gene regulatory function.