The binding of myristoylated N-terminal nonapeptide from neuron-specific protein CAP-23/NAP-22 to calmodulin does not induce the globular structure observed for the calmodulin–nonmyristoylated peptide complex

Abstract

CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is N^α-myristoylated and interacts with calmodulin (CaM) in the presence of Ca²⁺ ions. Takasaki et al. (1999, J Biol Chem 274:11848–11853) have recently found that the myristoylated N-terminal nonapeptide of CAP-23/NAP-22 (mC/N9) binds to Ca²⁺-bound CaM (Ca²⁺/CaM). In the present study, small-angle X-ray scattering was used to investigate structural changes of Ca²⁺/CaM induced by its binding to mC/N9 in solution. The binding of one mC/N9 molecule induced an insignificant structural change in Ca²⁺/CaM. The 1:1 complex appeared to retain the extended conformation much like that of Ca²⁺/CaM in isolation. However, it could be seen that the binding of two mC/N9 molecules induced a drastic structural change in Ca²⁺/CaM, followed by a slight structural change by the binding of more than two but less than four mC/N9 molecules. Under the saturated condition (the molar ratio of 1:4), the radius of gyration (R_g) for the Ca²⁺/CaM-mC/N9 complex was 19.8 ± 0.3 Å. This value was significantly smaller than that of Ca²⁺/CaM (21.9 ± 0.3 Å), which adopted a dumbbell structure and was conversely 2–3 Å larger than those of the complexes of Ca²⁺/CaM with the nonmyristoylated target peptides of myosin light chain kinase or CaM kinase II, which adopted a compact globular structure. The pair distance distribution function had no shoulder peak at around 40 Å, which was mainly due to the dumbbell structure. These results suggest that Ca²⁺/CaM interacts with N^α-myristoylated CAP-23/NAP-22 differently than it does with other nonmyristoylated target proteins. The N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins suggests that the protein myristoylation plays important roles not only in the binding of CAP-23/NAP-22 to Ca²⁺/CaM, but also in the protein–protein interactions related to other myristoylated proteins.