SxIP binding disrupts the constitutive homodimer interface of EB1 and stabilizes EB1 monomer.

SxIP is a microtubule tip localizing signal found in many +TIP proteins that bind to the hydrophobic cavity of the C-terminal domain of end binding protein 1 (EB1) and then positively regulate the microtubule plus-end tracking of EBs. However, the exact mechanism of microtubule activation of EBs in the presence of SxIP signaling motif is not known. Here, we studied the effect of SxIP peptide on the native conformation of EB1 in solution. Using various NMR experiments, we found that SxIP peptide promoted the dissociation of natively formed EB1 dimer. We also discovered that I224A mutation of EB1 resulted in an unfolded C-terminal domain, which upon binding with the SxIP motif folded to its native structure. Molecular dynamics simulations also confirmed the relative structural stability of EB1 monomer in the SxIP bound state. Residual dipolar couplings and heteronuclear NOE analysis suggested that the binding of SxIP peptide at the C-terminal domain of EB1 decreased the dynamics and conformational flexibility of the N-terminal domain involved in EB1-microtubule interaction. The SxIP-induced disruption of the dimeric interactions in EB1, coupled with the reduction in conformational flexibility of the N-terminal domain of EB1, might facilitate the microtubule association of EB1.