Figure 2: C-terminal binding region of XMAP-Ct is a bona fide coiled coil.

(a) Conformational chemical shift pattern by NMR of XMAP-Ct (pH 5.9 and 25 °C) as a function of the protein sequence suggests a helical propensity. Above: Cα (grey bars) and Cβ (white bars); below: comparison of the Cα conformational chemical shits of XMAP-Ct (white bars) and pCt (pH 6.1, 5 °C; grey bars). (b) A peptide derived from XMAP-Ct coiled-coil region was synthesized (pCt). Positions e and d of the heptad repeat were mutated to introduce either an opposite charge (pCt KD) or to break the hydrophobic interaction (pCt_LS). Scheme of a wheel diagram depicting the disposition of key residues in a simple two-helix coiled-coil interaction. A coiled-coil interaction of a more complex nature (trimer/tetramer) will also follow the same principle forming a hydrophobic core with the charged residues pointing outwards. (c) ITC and SPR binding curves showing TD4 interaction with pCt (red) and no interaction with pCt_KD (blue). The calculated K_D values were 11 and 30 μM by ITC and SPR, respectively. (d) Western blot of pull-downs of CSF, XMAP-Ct and its mutants. Only XMAP-Ct and mutants of conserved charge (XMAP-Ct_KR and XMAP-Ct_LI) can pull down XTACC3, whereas by reversing the charge (XMAP-Ct_KD and XMAP-Ct_LS) mutants were unable to pull down XTACC3. The efficiency of the pull-down of the tagged proteins was verified by Coomassie blue staining. RU, response unit.