Figure 5: The C-cap motif of the high affinity DARPins is mobile in the crystal structure.

(a) The H118R mutation destabilizes the C-cap. The TM-3 structure (blue) was superimposed to uncomplexed D1, colored as in Fig. 4a. The Arg118 side chain adopts two alternate conformations in TM-3 (Supplementary Fig. 6). Both would clash with the C-cap if it were folded as in D1 (3 distances shorter than 1.5 Å are highlighted as black solid lines), as would the many other Arg118 conformations we modeled. (b) Comparison of the complexes of tubulin (beige) with D1 (green) and with A-C2 (blue). The β tubulin subunits have been superimposed. Some side chains are drawn to help visualize the shift between the DARPins. (c) View of the tubulin−D1 interface colored by electrostatic potential. (Left) Electrostatic potential surface of β tubulin (red, negative; blue, positive) with bound D1 (green). The side chains of two acidic residues of the C-cap that are close to an acidic part of the surface of tubulin are shown. The Cαs of residues 148 and 149 are also highlighted as magenta spheres. Subtilisin cleaves the affinity-improved DARPins preferentially after these two residues (see text and Supplementary Fig. 2). (Right) Electrostatic potential surface of D1 centered on the tubulin-interacting surface. Note that the two panels are not to scale.