Electrostatic buffering of non-native interactions in the transition state ensemble for binding of intrinsically disordered proteins

Intrinsically disordered protein regions (IDRs) mediate key steps in cellular signaling but the nature of the energy barriers crossed by IDRs upon association and the energetic features of the transition state ensemble (TSE) for binding remain elusive. Short linear motifs (SLiMs) are small functional units found within IDRs that fold upon binding to globular domains. Here, we use the LxCxE SLiM from the human papillomavirus E7 protein (LxCxEWT) binding to the retinoblastoma (Rb) protein as a minimal model system for an IDR-domain interaction. We combine extensive mutagenesis with electrostatic dissection to gain information on the binding TSE energetics and rule out ground state effects using Far-UV CD and NMR. This approach uncovers strong compensatory energetics whereby stabilizing electrostatic interactions play a key role buffering multiple weakly destabilizing non-native interactions in the binding TSE for LxCxEWT. Electrostatic buffering may enable a dynamic search for native contacts and fine tuning of the TSE energetics. A global analysis of 177 mutations reveals non-native energetics in the binding TSE of many IDRs, suggesting that electrostatic buffering may be a widespread phenomenon that dictates functional selection for charge content within IDRs.