Supplementary Figure 6: Characterization of the CKK MT-binding site; energetics and electrostatic contributions.

(a) CAMSAP3 residue-wise variation in solvent accessible surface area (top, black bars) and energetic contributions to MT binding (bottom, green bars). Selected CKK residues showing major solvent accessibility changes and energetic contributions are labelled in green, with corresponding interacting tubulin residues labelled in light grey for α-tubulin and dark grey for β-tubulin. Red stars indicate the location of residues with ssNMR signal shifts. Energetic contributions were determined from molecular dynamics simulations using the Amber MMGBSA routine. Data represent mean ± SD. Solvent accessibility was determined with Bio3D (Grant, B.J. et al., Bioinformatics 22, 2695-2696, 2006, see Methods).

(b) Surface representation of the MT-CKK interface on tubulin colored by electrostatic potential, with the CKK binding site indicated by a green dashed outline. The surface was calculated with the APBS (Baker, N.A. et al., PNAS 98, 10037-10041, 2001) and pdb2pqr (Dolinsky, T.J. et al., Nucleic acids research 35, W522-525, 2007).

(c) The corresponding front view of the surface-represented CKK (left) colored in the same way as panel b, while a 180° rotation of this view illustrating the tubulin-facing surface of the CKK is shown on the right.

(d) MT pelleting assay, in which the CAMSAP3 CKK and MTs were mixed in equimolar ratios at different salt concentrations in BRB35 buffer. As controls, the CKK domain and MTs were processed alone. The supernatants (S) and pellets (P) were separated on a 15 % SDS-PAGE and stained with Coomassie.

(e) Tubulin-facing surface representation of CAMSAP3 CKK surface showing the residue conservation between the M. musculus, C. elegans, D. melanogaster CKK domains. Dark green, invariant residues; light green, similar residues; white, non-conserved residues. Equivalent view to right hand image in panel (c) is shown.