Moesin binds to microtubules via its N-terminal FERM domain. (A) A schematic depicting the three domains of Moesin. D.m, Drosophila melanogaster. (B–D) Microtubule cosedimentation assay. Samples from protein alone (−MT) and protein with microtubules (+MT) are shown in Coomassie blue–stained gels. S, supernatant; P, pellet. (B) Sedimentation assay with purified domains of Moesin. (C) FERM domain was preincubated with GST-CERMAD of wild-type Moesin (residues 483–578; top) or with GST-CERMAD of Moesin483–559 (residues 483–559; bottom) before performing microtubule cosedimentation. (D) Untreated or subtilisin-treated microtubules were tested in sedimentation assay with Moesin1–559 or FERM of Moesin. (E) A crystal structure of FERM (blue) and CERMAD (red) complex of Moesin. Candidate lysine residues (K238, K212, and K213) are highlighted in green. (F) Sequence alignment of the microtubule binding region of ERM proteins and Merlin. H.s, H. sapiens. (G) Comparison of binding affinities to microtubules between FERM wild type and K212,213M. (H) Fractions of microtubule-bound Moesin FERM from three independent microtubule sedimentation experiments were plotted against microtubule concentrations and fitted to a hyperbola to determine the Kd. Error bars represent SDs.
