NMR Structure Reveals Novel Interactions between Intrinsically Disordered PEP-19 and Calmodulin

Abstract

Background: PEP-19 has no known intrinsic activity other than binding predominately to the C-domain of calmodulin (C-CaM), yet it is implicated in numerous cellular processes. We showed that an acidic sequence in PEP-19 is required to greatly increase the rates of Ca2+ binding to C-CaM. Importantly, the acidic sequence is also required for PEP-19 to sensitize HeLa cells to ATP-induced Ca2+ release. Goal: The goal of the current study was to determine the high-resolution NMR solution structure of the PEP-19/apo C-CaM complex.Results: Apo C-CaM adopts a semi-open conformation when bound to PEP-19, with the helices E and F of Ca2+ binding site III showing the greatest change in angle relative to free apo C-CaM. The conformation of Ca2+ binding loop III is similar to that in free apo C-CaM, but loop IV adopts a different conformation with increased conformational exchange when bound to PEP-19. Residues 1-29 in PEP-19 remain disordered when bound to C-CaM, and are thus accessible for potential interactions with other proteins. The IQ motif in PEP-19 adopts a well-defined alpha helix that binds to a hydrophobic groove in apo C-CaM. The C-terminal part of the acidic sequence is alpha helical, but the N-terminal portion (aa 28-36) forms loop and coil structures that are stabilized by interactions between Ile32 and Met34 in PEP-19 and hydrophobic residues in Ca2+ binding site III of C-CaM. This allows acid side chains in PEP-19 to extend toward the solvent to greatly increase negative charge density near site III of C-CaM.Conclusions: The structure suggests that the acidic sequence in PEP-19 modulates Ca2+ binding to site III of C-CaM by direct interactions and/or electrostatic steering of Ca2+, but that allosteric effects increase conformational exchange to modulate Ca2+ binding to site IV.