Figure 5. Effect of point mutations in ATi3N or ATctN on their interactions with CaM.

A. Modeled structures of CaM-ATi3(214–231) and CaM-ATct(302–317). The complexes of CaM and CaM binding motif in the i3 loop ATi3(214–231) or the carboxyl terminal tail ATct(302–317) of the receptor were modeled as described in Experimental Procedures. The target peptides are colored in red. Residues W219 in ATi3(214–231)−SYTLIWKALKKAYEIQKN, and F309 and F313 in ATct(302–317)−YGFLGKKFKKYFLQLL are displayed with sticks and are colored in blue. Calcium atoms are shown as orange spheres. The N- and C- termini of CaM are also labeled. Helices and sheets in CaM are colored in green and yellow, respectively. B. Effect of point mutations at ATi3N or ATctN on their interaction with CaM. 50 pmol of wild type GST-fusion proteins including GST-ATi3N(213–234) and GST-ATctN (297–324), and 50 pmol of mutated GST-fusion proteins including GST-ATi3N(W219A), GST-ATctN(F309A) and GST-ATctN(F313A), were incubated with purified bovine brain CaM in a buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl₂. The protein complexes were pulled down by gluthathione-sepharose 4B beads, and subjected to immunoblot with a specific anti-CaM antibody. GST-fusion proteins were visualized in the gels by Coomassie blue staining (the lower gel panel). The summary graph represents relative densities of the ratio of the CaM in the immunoblots and the loaded GST-fusion proteins as determined by Coomassie blue staining. The bars represent mean ± S.E. from 5 independent experiments. * or # stand for P<0.01 as compared with wild type GST-fusion proteins, respectively.