Calmodulin (CaM) plays a vital role in calcium homeostasis by allosterically modulating intracellular calcium channels including the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1). Apo (calcium-free) CaM activates hRyR1 while calcium-saturated CaM inhibits it. Two CaM-binding regions (residues 1975–1999 and 3614–3643) identified in each RyR1 monomer were proposed to allow CaM to bridge adjacent RyR1 subunits. We explored the distinct roles of CaM domains by using fluorescence anisotropy to determine the affinity of CaM1–148 (full-length), CaM1–80 (N-domain) and CaM76–148 (C-domain) for peptides encompassing hRyR1 residues 1975–1999 or 3614–3643. Both CaM1–148 and CaM76–148 associated in a calcium-independent manner with similar affinities for hRyR1(3614–3643)p while CaM1–80 required calcium and bound ~250-fold more weakly. Association of CaM1–148, CaM1–80 and CaM76–148 with hRyR1(1975–1999)p was much less favorable than with hRyR1(3614–3643)p; differences between the two CaM domains were smaller. Equilibrium calcium titrations monitored by steady-state fluorescence demonstrated that both hRyR1 peptides increased the calcium-binding affinity of both CaM domains. These thermodynamic properties support a prior model in which the CaM C-domain associates with RyR1(3614–3643) at low levels of calcium, positioning CaM to rapidly respond to calcium efflux. However, the affinity of the N-domain of CaM for hRyR1(1975–1999)p is insufficient to explain a model in which CaM bridges adjacent RyR1 subunits within the tetramer. This indicates that other protein factors or properties of the tertiary or quaternary structure of hRyR1 contribute to the energetics of CaM-mediated regulation.

Human voltage-gated sodium channel NaV1.2 has a single pore-forming α-subunit and two transmembrane β-subunits. Expressed primarily in the brain, NaV1.2 is critical for initiation and propagation of action potentials. Milliseconds after the pore opens, sodium influx is terminated by inactivation processes mediated by regulatory proteins including calmodulin (CaM). Both calcium-free (apo) CaM and calcium-saturated CaM bind tightly to an IQ motif in the C-terminal tail of the α-subunit. Our thermodynamic studies and solution structure (2KXW) of a C-domain fragment of apo 13C,15N-CaM (CaMC) bound to an unlabeled peptide with the sequence of rat NaV1.2 IQ motif showed that apo CaMC (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaMC. However, we could not monitor the NaV1.2 residues directly, and no structure of full-length CaM (including the N-domain of CaM (CaMN)) was determined. To distinguish contributions of CaMN and CaMC, we used solution NMR spectroscopy to assign the backbone resonances of a complex containing a 13C,15N-labeled peptide with the sequence of human NaV1.2 IQ motif (NaV1.2IQp) bound to apo 13C,15N-CaM or apo 13C,15N-CaMC. Comparing the assignments of apo CaM in complex with NaV1.2IQp to those of free apo CaM showed that residues within CaMC were significantly perturbed, while residues within CaMN were essentially unchanged. The chemical shifts of residues in NaV1.2IQp and in the C-domain of CaM were nearly identical regardless of whether CaMN was covalently linked to CaMC. This suggests that CaMN does not influence apo CaM binding to NaV1.2IQp.

Human voltage-gated sodium (Na) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane β-subunits. Na pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all Na isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of Na1.2, Na1.5, and Na1.6 showed that CaM binding was mediated by the C-domain of CaM (CaM), while the N-domain (CaM) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other Na isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of Na1.1, Na1.4, and Na1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaM; resonances of CaM were identical to free CaM. Thus, CaM residues contribute to the interface with the IQ motif, while CaM is available to interact elsewhere on the channel.

Tcb2 is a putative calcium-binding protein from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila. It has been hypothesized to participate in several calcium-mediated processes in Tetrahymena, including ciliary movement, cell cortex signaling, and pronuclear exchange. Sequence analysis suggests that the protein belongs to the calmodulin family, with N- and C-terminal domains connected by a central linker, and two helix-loop-helix motifs in each domain. However, its calcium-binding properties, structure and precise biological function remain unknown. Interestingly, Tcb2 is a major component of unique contractile fibers isolated from the Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an ATP-independent type of contraction. Here we report the (1)H, (13)C and (15)N backbone and side-chain chemical shift assignments of the C-terminal domain of the protein (Tcb2-C) in the absence and presence of calcium ions. (1)H-(15)N HSQC spectra show that the domain is well folded both in the absence and presence of calcium, and undergoes a dramatic conformational change upon calcium addition. Secondary structure prediction from chemical shifts reveals an architecture encountered in other calcium-binding proteins, with paired EF-hand motifs connected by a flexible linker. These studies represent a starting point for the determination of the high-resolution solution structure of Tcb2-C at both low and high calcium levels, and, together with additional structural studies on the full-length protein, will help establish the molecular basis of Tcb2 function and unique contractile properties.

Na V 1.2 EFL domain allosterically enhances Ca 2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD Graphical abstract Highlights d In solution, only the IQ motif of Na V 1.2 CTD(1,777-1,937) contacts CaM ± Ca 2+ d Ca 2+ changes interfaces of CaM + Na V 1.2-IQ p and CaM + Na V 1.2-CTD complexes identically d Ca 2+ binds to N-domain sites of CaM + Na V 1.2-CTD preferentially relative to free CaM d Apo pathogenic CaM mutants bind the Na V 1.2 CTD as well or better than WT CaM