Gerendasy D D, Herron S R, Jennings P A, Sutcliffe J G
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1995 Mar 24;270(12):6741-50. doi: 10.1074/jbc.270.12.6741.
Two neuronal protein kinase C substrates, RC3/neurogranin and GAP-43/neuromodulin, preferentially bind to calmodulin (CaM) when Ca2+ is absent. We examine RC3.CaM and GAP-43.CaM interactions by circular dichroism spectroscopy using purified, recombinant RC3 and GAP-43, sequence variants of RC3 displaying qualitative and quantitative differences in CaM binding affinities, and overlapping peptides that cumulatively span the entire amino acid sequence of RC3. We conclude that CaM stabilizes a basic, amphiphilic alpha-helix within RC3 and GAP-43 under physiological salt concentrations only when Ca2+ is absent. This provides structural confirmation for two binding modes and suggests that CaM regulates the biological activities of RC3 and GAP-43 through an allosteric, Ca(2+)-sensitive mechanism that can be uncoupled by protein kinase C-mediated phosphorylation. More generally, our observations imply an alternative allosteric regulatory role for the Ca(2+)-free form of CaM.
两种神经元蛋白激酶C底物，即RC3/神经颗粒素和GAP-43/神经调节蛋白，在无Ca2+时优先与钙调蛋白（CaM）结合。我们使用纯化的重组RC3和GAP-43、在CaM结合亲和力上表现出定性和定量差异的RC3序列变体，以及累积覆盖RC3整个氨基酸序列的重叠肽段，通过圆二色光谱法研究RC3.CaM和GAP-43.CaM的相互作用。我们得出结论，仅在无Ca2+时，CaM在生理盐浓度下稳定RC3和GAP-43内的一个碱性两亲性α螺旋。这为两种结合模式提供了结构证实，并表明CaM通过一种变构的、Ca(2+)敏感机制调节RC3和GAP-43的生物学活性，该机制可被蛋白激酶C介导的磷酸化作用解偶联。更普遍地说，我们的观察结果暗示了无Ca2+形式的CaM具有一种替代的变构调节作用。
