Figure 3:

Phage display screening identifies peptides that bind MSN at distinct sites. (A) Sequences of phage display peptides. Underlined region corresponds to the variable region within the phage-derived sequence. (B) Crystal structure of FERM domain of MSN bound to the C3P-pd peptide. FERM subdomains are displayed as in 1B. C3P-pd peptide (magenta) binds in a pocket between the F1 and F3 subdomains. (C) Close-up view of C3P-pd peptide bound to MSN. Peptide intramolecular H-bonds are shown (purple dashed lines). Sidechains of MSN residues interacting with the peptide are displayed, together with their bonding to the peptide (gold dashed lines). (D) Superimposed F3 subdomains of MSN from apo (cyan) and C3P-pd bound (light yellow) structures. C3P-pd peptide binding causes a 2.3 Å movement of the beta sheet away from the alpha helix in the MSN F3 lobe, measured from H288 and R246 carbonyl carbon atoms. (E) Crystal structure of FERM domain of MSN bound to both C3P-pd and C3S1-pd peptides. (F) Close-up of C3S1-pd binding to F3 lobe of MSN. H-bond contacts are shown (green dashed lines). (G) Space-filling model of MSN FERM domain, showing C3P-pd (magenta) and CD44 (cyan) binding relative to proposed PIP₂ binding pocket (PIP₂-BP; blue positively-charged surface). (H, I, J) MSN TR-FRET inhibition assays, with acceptor fluorophore conjugated to either (H) CD44, (I) C3P-pd, or (J) C3S1-pd peptides. Unlabelled peptides were used as competitors (C3S1-pd, blue circle; C3P-pd, red square; CD44, green triangle). (K) IC₅₀ values derived from H, I, and J. Asterisk denotes stimulatory rather than inhibitory values (L) Binding properties MSN association to C3P-pd and C3S1-pd peptides, measured by ITC. See Figure S3 for thermograms and corresponding fitted curves. K_D=dissociation constant, N=stoichiometry, ΔH=enthalpy.