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

Abstract

End Binding protein 1 (EB1) is a key component of the signalling networks located at the plus ends of microtubules. It incorporates an N-terminal microtubule binding CH domain and the C-terminal EBH domain that interacts with the SxIP-containing sequences of other microtubule plus end tracking proteins (+TIPs). By using a series of SxIP containing peptides derived from the microtubule-actin cross-linking factor, MACF, we show that the SxIP motif itself binds to EBH with low affinity, and the full interaction requires contribution of the post-SxIP residues. Based on the solution structure and dynamics of the EBH/MACF complex we proposed 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, which subsequently induces folding of the unstructured C-terminus and transition to the stable complex. We dissect contributions from different interactions 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 of small molecule inhibitors that can block SxIP interaction with EB1.