Structure of calmodulin complexed with an olfactory CNG channelfragment and role of the central linker: Residual dipolar couplingsto evaluate calmodulin binding modes outside the kinase family

Abstract

The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs.

Structural Insights into Calmodulin-regulated L-selectin Ectodomain Shedding

“…Consequently, RDC analysis can be used to determine the orientation of the two lobes of CaM when bound to LSELl and provide a homologous starting model for the structure of CaM in the complex (18,35). Because RDCs are a function of bond vector orientation in relation to the external magnetic field, they are particularly useful for orienting two domains of a protein with respect to each other (36).…”

Calmodulin Regulates Ca2+-sensing Receptor-mediated Ca2+ Signaling and Its Cell Surface Expression

“…RDC has been used to assist in homology modeling, determine relative domain orientations in multidomain proteins, and characterize molecular binding processes (25,26). This recently developed technique has been applied to investigate the complex mode of calmodulin and its target peptide/fragments (26,27).…”

“…This recently developed technique has been applied to investigate the complex mode of calmodulin and its target peptide/fragments (26,27). To assess the binding mode of CaM and the CaSR utilizing this approach, we compared the measured RDCs with calculated values from structures deposited in the Protein Data Bank (PDB) (listed in supplemental Table S1).…”

Structural Determinants of Calmodulin Binding to the Intracellular C-terminal Domain of the Metabotropic Glutamate Receptor 7A

“…The combined RDC and chemical shift data provide good evidence for a classic wraparound structure of the Ca 2ϩ /CaM⅐mGluR 7A complex. Residual dipolar couplings have been used successfully to classify CaM-binding motifs present in the protein kinase family (48) and, more recently, to evaluate motifs outside the kinase family (47). The latter proved to be successful only if the RDCs used in the analysis were restricted to those residues located in structured regions of CaM.…”