Site-Specific Methionine Oxidation and Calmodulin Molecular Dynamics

Abstract

Calmodulin (CaM) is a prototypical and versatile Ca2þ sensor. CaM is present in all eukaryotic cells, mediating Ca2þ-dependent signaling. Upon binding Ca2þ, CaM changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on CaM, little is known on how target proteins regulate CaM's ability to bind Ca2þ. Recently we identified a novel splice variant (KCNN2-b) of KCNN channels which shows different Ca2þ sensitivity compared to the original KCNN2-a. KCNN2-b has three additional amino acid residues in the CaM binding domain (CaMBD). We take advantage of the CaMBDs from these splice variants to investigate their impact on CaM. Crystal structures show that interactions with these KCNN2 splice variants force CaM to adopt different conformations and thus change its affinity for Ca2þ at its C-lobe. All four EF-hands of CaM in the CaM-CaMBD2-b complex are occupied by Ca2þ ions, whereas in the CaM-CaMBD2-a complex only EF-hands at CaM N-lobe are loaded. Experiments with mutant CaMs support this difference in Ca2þ binding stoichiometry. Mutations of the EF hands at CaM N-lobe (CaM12) almost abolished the interaction with both CaMBDs. The mutant CaM unable to bind Ca2þ at C-lobe (CaM34) interacts with CaMBD2-a substantially, with a similar amplitude compared to wild type CaM. The thermodynamic profile of CaM34-CaMBD2-b, however, is distinct from that of the wild type CaM-CaMBD2-b. This difference in metal ion binding stoichiometry is also proven in solution by Tb3þ Fluorescence measurements. We further manipulated the three residues insertion by shortening it to two residues (CaMBD2-AlaAla) or one residue (CaMBD2-Ala). Different metal ion binding stoichiometry and Ca2þ sensitivity was observed between CaM complexes with these two mutant CaMBDs, which furthers our understanding on the fine-tuning of CaM's affinity for Ca2þ by target proteins.