An Acidic Sequence in Neurogranin is Required to Modulate Ca2+ Binding to Calmodulin

Abstract

Background: Neurogranin (Ng) and PEP-19 are small proteins with no known intrinsic activity other than binding to calmodulin (CaM) via their IQ motifs, yet they have been implicated in numerous normal and pathological processes. We showed that PEP-19 is intrinsically disordered, and that an acidic sequence adjacent to its IQ motif is required for PEP-19 to modulate Ca2+ binding to CaM, and to sensitize HeLa cells to ATP-induced Ca2+ release. Goals: Ng has an acidic sequence, but with significantly different composition than PEP-19. Thus, the goals of the current study were to determine: 1) If the acidic sequence in Ng is required to modulate Ca2+ binding to CaM; and 2) Use NMR to compare the effects of Ng derivatives on the conformation of CaM. Results: Ng greatly increases the Ca2+ koff at the C-domain of CaM, but has little effect on the kon, thereby decreasing Ca2+ binding affinity. The peptide Ng(29-49), which includes only the consensus IQ motif does not increase the Ca2+ koff, but Ng(13-49), which includes the acidic region of Ng mimics the effect of intact Ng on Ca2+ binding to CaM. The 1H 15N HSQC spectra of Ng show it to be an intrinsically disordered protein. Effects of Ng protein and peptides on the NMR spectra of CaM are consistent with their relative effects on Ca2+ binding. Also, effects of Ng (29-49) on amide chemical shift perturbations and backbone dynamic properties of CaM are different from Ng or Ng(13-49), especially in the apo state. Conclusions: Modulating Ca2+ binding to CaM relies on the acidic region of both PEP-19 and Ng. Tuning Ca2+ mobilization pathways by PEP-19 and Ng expands the biological significance of these intrinsically disordered regulators of CaM signaling.