Alternative Splicing of a Structured Partner Alters the Folding-Upon-Binding Trajectory of an Intrinsically Disordered Protein.

Folding-upon-binding of intrinsically disordered proteins (IDPs) is governed by a complex interplay of kinetic and thermodynamic factors shaped by the structure and conformational dynamics of both binding partners. Alternative splicing offers a natural way to remodel the conformational energy landscape of structured partners, yet how such biologically relevant changes influence the molecular recognition trajectories of interacting IDPs remains poorly understood. Here, using the small GTPase Rac1 and its oncogenic splice variant Rac1b as a model system, we integrate X-ray crystallography, isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy to investigate how the 19-residue insertion in Rac1b alters recognition of the disordered signaling effector POSH. We show that the insertion restricts POSH to partial folding-upon-binding and determine the crystal structure of the POSH-Rac1b complex at 1.77 Å resolution. The structure reveals that POSH stabilizes the otherwise dynamic switch regions of Rac1b in a signaling-competent conformation, while the insertion itself remains dynamic. NMR exchange experiments further delineate the molecular recognition trajectory of POSH upon binding to Rac1b, revealing a folding intermediate characterized by a 5.7-fold slower association rate and a 3-fold faster dissociation rate compared to Rac1. Together, these results demonstrate that the insertion, kinetically and entropically, destabilizes the effector-bound state of Rac1b, directly linking enhanced conformational dynamics to impaired downstream signaling. More broadly, our work illustrates how alternative splicing of folded proteins can reshape folding trajectories, binding kinetics, and thermodynamic landscapes of IDP-mediated interactions, thereby rewiring cellular signaling networks.