Residues that follow SxIP motif enhance binding by engaging with the C-terminus of the EB1 EBH domain.

(A) Crystal structure of the EBH domain in the complex with MACF peptide (PDB ID 3GJO). EB1 is shown as a cartoon with the subunits of the dimer coloured in green and cyan, and a semi-transparent surface. The MACF peptide is coloured orange, with the SxIP motif chains-chains shown as sticks. The zoomed region highlights the binding pocket of EBH formed by the surface of the coiled-coil and the folded C-terminus. (B) Zoom on the partly formed SxIP binding pocked in the structure EBH domain free in solution (PDB ID 3EVI) in the same orientation as (A). The C-terminal region is unfolded. (C) EB1 chemical shift changes in the 1H,15N-HSQC spectra induced by 4MACF (blue), 6MACF (green) and 11MACF (red) peptides. (D) Relative intensities of cross-peaks in the 1H,15N-HSQC spectra of the EBH free in solution (black) and in the presence of 4MACF (blue), 6MACF (green) and 11MACF (red) peptides. (E) “Dock and Lock” binding model that explicitly considers the role of EBH C-terminus in the interaction with the SxIP peptide. Initially, the binding pocket is partially formed and only contains SxIP-recognition region. Following the initial binding (“Dock”), the post-SxIP region of the peptide induces the folding of the of the C-terminus and formation of the full binding pocket (“Lock”). The deletion of the EBH C-terminus removes the “Lock” stage of the binding, thus reducing the affinity of the interaction.

Binding parameters of the EBH domain interactions with peptides

Structure and dynamics of the EB1 EBH domain.

(A) Superposition of 20 NMR structures calculated for EBH in the complex with 11MACF peptide (left). Previously reported NMR structures of the free EBH (PDB ID 6EVI, middle) and the EBH complex with a small SxIP-like molecule 1a (PDB ID 6EVI, right) are shown for comparison. The EBH subunits are coloured in green and cyan, the peptide and the small molecule in orange and magenta. (B) Representation of the residues forming the contact interface between the EBH and 11MACF peptide, both shown as cartoon, with side chain displayed for all the residues involved in the contacts between the two molecules. EB1 is coloured in grey and 11MACF in green, with oxygen shown in red and nitrogen shown in blue. (C) Order parameters S2 (left) and exchange contributions into the relaxation rate Rex (right) calculated from the relaxation parameters for the free EBH (top) and EBH in complex with 11MACF peptide (bottom) mapped on the EBH solution structure. The thickness of the tube is proportional to the value of the corresponding parameter.

Enhancement of the peptide binding through the substitution of the post-SxIP residues.

(A) Folding of the C-terminus brings the hydrophobic EBH residues of that region into the contact with the TPQ region of the peptide. Structure of the free EBH (left) and EBH in the complex with 11MACF (right). (B) Proximity of the positively charged RK residues of the peptide to the negatively charged patch on the EBH surface. The peptide is shown as a cartoon, RK side-chains are shown in a stick representation. Positive and negative electrostatic surface potential is represented by blue and red colour, respectively. (C) The ITC titration (top) and the binding isotherm fitted into a single-site binding model (bottom) of the ITC binding experiments for the 11MACF peptide and the mutants 11MACF-LLL, 11MACF-VLL and 11MACF-VLLRK. The peptide sequences are shown above the graphs, with the changed regions highlighted by the red (mutation) and green (insertion) colours. (D) The thermodynamic parameters (ΔG (green), ΔH (blue) and -TΔS (red) calculated from ITC data.

NMR data show large decrease in the exchange rates on the mutations of the post-SxIP residues.

(A) 11MACF-LLL and 11MACF-VLL peptides induce increased chemical shift changes compared to the 11MACF. Chemical shift differences in the 1H,15N-HSQC spectra between the free EBH and the EBH/11MACF complex (blue), EBH/11MACF and EBH/11MACF-LLL (orange), and EBH/11MACF and EBH/11MACF-VLL (green). (B) Chemical shift changes in the 1H,15N-HSQC spectra on peptide addition observed for the EBH interactions with different peptides illustrated for the Thr249 signal. Superposition of the spectra for the titration of the EBH-ΔC with 11MACF (left), EBH with 11MACF (middle), and EBH with 11MACF-VLL (right). Signals of the free EBH are shown in red, fully bound EBH in blue, and the intermediate titration points are shown in the pale colours. Notice two additional signals that are observed for the EBH/11MACF-VLL titration at the intermediate concentrations corresponding to the non-symmetrical form where EBH dimer binds a single peptide. These signals can only be observed when the exchange between the different forms is very slow. (C) Example of the CEST profiles measured at the irradiation field strength of 12.5, 25 and 50 Hz (left to right) for Asp250. Solid curve represents the fitting of the data into the global exchange model with the dissociation rate 130 s-1 calculate with Chemix software. (D) Exchange rates calculated for the EBH interaction with 11MACF peptide from the combination of the NMR data using the two-stage interaction model, where the folding of the EBH C-terminus follows the peptide binding. (E) Free energy contributions into the EBH interaction with the 11MACF peptide (left) and the mutated 11MACF-VLL peptide (right). The SxIP motif itself contributes approximately half of the binding energy (-15.9 kJ/mol), with the second half created by the interaction of the TPQRK region with the coiled-coli folded part of EBH (-9.3 kJ/mol), and the C-terminal EBH region that folds on the peptide binding (-6 kJ/mol). The VLL mutation increases the interaction with both the coiled-coli folded part of EBH energy (-28.4 kJ/mol) and the EBH C-terminus energy (-12.1 kJ/mol).

High affinity peptides form comet-like structures that colocalise with EB1 comets.

(A) Live images of HeLa cells transiently transfected with SxIP peptide constructs and EB1 plasmid. Images represent averaging three frames at 1 second intervals. Inserts represent zoomed-in regions of the cell to depict SxIP peptides at EB1 comets. Scale bar; 5μM. (B) Ratio of SxIP comets to EB1 comets present in a cell. (C) EB1 comet number per cell. Lines represent the mean with error bars representing the standard deviation. Changes in comet number with each peptide were not significant when T-test was used with the EB1 construct only control (EB1). All experiments were performed to an N=3.