Dock-and-lock binding of SxIP ligands is required for stable and selective EB1 interactions.

End binding protein 1 (EB1) is a key component of the signalling networks at microtubule plus ends. It contains an N-terminal microtubule-binding CH domain and a C-terminal EBH domain interacting with SxIP-containing sequences of other microtubule plus end tracking proteins (+TIPs). Using a series of SxIP-containing peptides derived from the microtubule-actin cross-linking factor (MACF), we demonstrate that the SxIP motif itself binds to EBH with low affinity, and that the full interaction requires contribution of residues following the SxIP motif. Based on the solution structure and dynamics of the EBH/MACF complex, we propose a two-step 'dock-and-lock' model for the EBH interaction with targets, where the SxIP motif initially binds to a partially formed EBH pocket. This subsequently induces folding of the unstructured C-terminus and transition to a stable complex. We dissect contributions into the binding and design MACF mutations of the post-SxIP region that enhance the affinity by two orders of magnitude, leading to a nanomolar interaction. We verify the enhanced recruitment of the mutated peptide to the dynamic plus ends of MTs in a live-cell experiment. Our model explains EB1's interaction with the SxIP-containing ligands and can be used to design small-molecule inhibitors blocking SxIP interaction with EB1.