Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of CaV1.2

Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca2+ channel (CaV1.2) regulates Ca2+ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with CaV1.2 under low resting [Ca2+], but is poised to change conformation and position when intracellular [Ca2+] rises. CaM binding Ca2+, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A1588, and C1614 and the IQ motif studied as overlapping peptides IQ1644 and IQ′1650 as well as their effect on calcium binding. (Ca2+)4-CaM bound to all four peptides very favorably (Kd ≤ 2 nM). Linkage analysis showed that IQ1644-1670 bound with a Kd ~ 1 pM. In the pre-IQ region, (Ca2+)2-N-domain bound preferentially to A1588, while (Ca2+)2-C-domain preferred C1614. When bound to C1614, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.

Highlights► Domains of CaM interact cooperatively when bound to sites “A”, “C” and “IQ”. ► Residues upstream of consensus IQ motif are critical for CaM N-domain interactions. ► CaV1.2 site “A” binds the Ca2+-CaM N-domain more favorably than Ca2+-CaM C-domain. ► A model is proposed for (Ca2+)4-CaM bridging a hairpin of CaV1.2 sites “A” and “C”.