Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy.
The interaction of Ca(2+)-free calmodulin (apoCaM) with the IQ motif corresponding to the calmodulin-binding domain of neurogranin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of uncomplexed apoCaM and apoCaM in complex with the IQ motif recorded at 750 MHz were studied and the backbone assignments of the protein in both forms were obtained by triple-resonance multidimensional NMR experiments. Chemical shift perturbations were used to map the binding surfaces. Only a single set of resonances was observed throughout the titration, indicating that the binding interaction is under fast exchange. Analysis of chemical shift changes indicates that (a) the main interaction and conformational changes occur in the C-terminal domain of calmodulin and (b) linker-1 (residues 40-44) between EF-1 and EF-2, linker-3 (residues 112-117) between EF-3 and EF-4, and the end of the alpha-helix H (residues 145-148) may be involved in the binding process. The dissociation constant (K(d)), estimated by fitting the chemical shift changes against the IQ peptide concentration, ranged from about 1.2 x 10(-5) to 8.8 x 10(-5) M. This result demonstrates that the interaction falls into the weak binding regime.
Cui
Yanfang
Y
State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, The Chinese Academy of Sciences, Wuhan 430071, People's Republic of China. yfcui@wipm.ac.cn
Wen
Jian
J
Hung Sze
Kong
K
Man
David
D
Lin
Donghai
D
Liu
Maili
M
Zhu
Guang
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D051579:Neurogranin; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013816:Thermodynamics
D001059:Apoproteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D009419:Nerve Tissue Proteins; D051579:Neurogranin; D002118:Calcium