Dose-response enhancement of 12.5 nM D-CaM fluorescence emission by the CaMBD.

<p><i>(A)</i> Effect of an incremental addition of the Q2AB (left column) or SK2 CaM binding domains (right column) in the emission spectra of 12.5 nM D-CaM both in the absence of free Ca²⁺ (top panels, 10 mM EGTA added) and in the presence of 3.9 µM free Ca²⁺ (bottom panels). The color of the traces changes from red to blue as the ligand concentration increases. <i>(B)</i> Relative concentration-dependent enhancement of 12.5 nM D-CaM fluorescence emission by SK2 in the presence (open circles) or absence (filled circles) of 3.9 µM Ca²⁺. The parameters used to fit a Hill equation to the data (continuous and dashed lines) were: Max = 122±4.3, EC₅₀ = 13.7±1.6 nM, h = 1.6±0.3 in absence of Ca²⁺, and Max = 123±1.4, EC₅₀ = 9.2±0.4 nM, h = 1.3±0.1 in the presence of Ca²⁺. The data represent the means ± standard error from three or more independent experiments. The error bars are smaller than the symbols. For comparison, the result of the fit of a Hill equation to the data for the effect of Q2AB of D-CaM fluorescent emission taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo2" target="_blank">[26]</a> is plotted in grey in the absence (continuous grey line) or presence of Ca²⁺ (dotted grey line). <i>(C)</i> Plot of the apparent binding affinity derived from the data in B obtained in absence (black column) or in presence of Ca²⁺ (white columns) for the proteins indicated. ***, significance at P≤0.001, *P≤0.05, unpaired Student’s t test.</p

Summary of the binding parameters obtained after fitting a one site Hill equation to the data in Fig. 4.

<p>See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone-0086711-g004" target="_blank">Fig. 4</a> for the experimental conditions used. h: Hill coefficient; IC₅₀: concentration producing 50% inhibition. Kd: affinity constant derived assuming a 1∶1 binding.</p

Summary of the binding parameters obtained after fitting a two sites Hill equation to the Q2AB data in Fig. 3.

<p>See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone-0086711-g003" target="_blank">Fig. 3</a> for the experimental conditions used. h: Hill coefficient; IC₅₀: concentration producing 50% inhibition.</p

The N-lobe binds preferentially to helix B in the absence of Ca²⁺, whereas the C-lobe binds preferentially to helix A in the presence of Ca²⁺.

<p>Competition curves with isolated CaM lobes (N or C) obtained using the individual CaM lobes and performed in the absence (filled symbols) or in the presence of Ca²⁺ (open symbols). D-CaM (12.5 nM) was mixed with helix A (hA, red symbols) or helix B (hB, black symbols) at a concentration corresponding to its calculated EC₅₀ for the increase in D-CaM fluorescence emission (46.4 and 65.6 nM in absence or presence of Ca²⁺ for helix A respectively, and 20.1 and 42.6 nM in absence or presence of Ca²⁺ for helix B respectively) and then each lobe was added incrementally at the concentrations indicated. The data represent the means ± standard error from three or more independent experiments, where some error bars were smaller than the symbols. The result of fitting a Hill equation to the competition curves is compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone-0086711-t003" target="_blank">Table 3</a>.</p

Q2AB weakens the Ca²⁺-CaM interaction.

<p><i>(A)</i> Relative increase in D-CaM fluorescence emission (12.5 nM) in response to increased Ca²⁺ concentrations in the presence (open circles) or absence (filled circles) of a molar excess of Q2AB (200 nM) or the indicated segment A mutants. We have previously shown that maximal D-CaM fluorescence is attained at this concentration for WT, L339R and R353G <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo2" target="_blank">[26]</a>. The lines are the result of fitting a Hill equation to the data. The data represent the means ± standard error from three or more independent experiments. Some error bars were smaller than the symbols. The EC₅₀ values obtained are (in µM): CaM = 0.72±0.02, CaM/Q2AB WT = 3.64±0.26, CaM/Q2AB R353G = 1.26±0.16, CaM/Q2AB L339R = 0.75±0.02. <i>(B)</i> Plot of the apparent binding affinity derived from the data in A. ***, significance at P≤0.001, *P≤0.05, unpaired Student’s t test.</p

The competition assay defines lobe specific interactions with SK2.

<p>Competition curves with isolated CaM lobes (N or C), with an equimolar mixture of N- and C-lobes (N&C) or with intact CaM (N–C). D-CaM (12.5 nM) was mixed with SK2 at a concentration corresponding to its calculated EC₅₀ for the increase D-CaM fluorescence emission (see Fig. 1 <i>C</i>, 9.2 and 13.7 nM in the presence or absence of Ca²⁺, respectively) and the competing peptides were added incrementally at the concentrations indicated. The data represent the means ± standard error from three or more independent experiments. The error bars are smaller than the symbols. The result of fitting a Hill equation to the competition curves is compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone-0086711-t001" target="_blank">Table 1</a>. <i>(A)</i> The effect of incremental addition of the lobes indicated obtained in the absence of Ca²⁺ (Left, 10 mM EGTA added) and in the presence of 100 µM free Ca²⁺ (right). <i>(B)</i> The effect of incremental addition of CaM WT (N−C) and of an equimolar mixture of the lobes (N&C) obtained in the absence of Ca²⁺ (left, 10 mM EGTA added) and in the presence of 100 µM free Ca²⁺ (right). (<i>C</i>) Comparison of the arithmetic addition of the curves obtained for each individual lobe (N+C) with the effect of an equimolar mixture (N&C) and with CaM (N−C) at concentrations under 200 nM in absence (left) or in the presence of 3.9 µM Ca²⁺ (that were indistinguishable from the results obtained in the presence of 100 µM Ca²⁺). <i>(D)</i> Plot of the reduction in fluorescence at the indicated concentration of competing lobe(s). ***, significance at P≤0.001, **P≤0.01, unpaired Student’s t test. <i>(E)</i> A model for the Ca²⁺-dependent CaM/SK2 interaction that can be derived from this set of experiments. 1. Both lobes cooperate in binding and the C-lobe, but not the N-lobe, is bound to SK2 in absence of Ca²⁺. 2. Ca²⁺ does not affect the interaction with the C-lobe. As the Ca²⁺ concentration increases the N-lobe becomes calcified. 3. The calcified N-lobe binds to SK2, leading to the observed increase in affinity in the presence of Ca²⁺. The data do not allow the oligomerization state to be established and therefore the dimerization of the CaM/SK2 complex that takes place upon Ca²⁺ binding is based on the resolved structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Schumacher1" target="_blank">[9]</a>.</p

Model for the Ca²⁺-dependent CaM/Q2AB interaction.

<p><i>(A)</i> Sequence alignment of segments A and B in which L339, R353 and S511 are underlined. The predicted secondary structure of Kv7.2 according to the GORV algorithm is indicated above the sequence (<a href="http://gor.bb.iastate.edu/" target="_blank">http://gor.bb.iastate.edu/</a>, h = alpha helix, e = extended, c = coiled). The circle beneath a residue indicates that it contacts the N-lobe, while those in contact with the C-lobe are indicated with a square, both of which are color coded according to the CaM surface contact. The contact surface area has been estimated using the Sobolev <i>et al.</i> algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Sobolev1" target="_blank">[52]</a>. <i>(B)</i> Interaction model. The Q2AB helices are depicted as rectangles, the CaM lobes as ovals. Binding to CaM is transient <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Mruk1" target="_blank">[23]</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-GomezPosada1" target="_blank">[24]</a>, and the interaction with helix A is critical for function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo1" target="_blank">[25]</a>. Given the greater affinity for helix B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Xu1" target="_blank">[22]</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo1" target="_blank">[25]</a>, it is more likely that CaM docks initially to this helix <i>via</i> the N-lobe, facilitating the interactions between the C-lobe and helix A. Subsequently, a dynamic equilibrium is established: 1.- In the absence of Ca²⁺ the N-lobe dominates the interaction and initially binds to helix B. 2.- Subsequently, the C-lobe engages, establishing an equilibrium between binding to helix A and helix B. 3.- In the presence of Ca²⁺ the C-lobe binds to the IQ site of helix A. 4.- The holo-N-lobe alternates between helix A and helix B. Upon calcification, the interaction between helix B and the N lobe is weakened and the binding between helix A and the C-lobe becomes more significant. Concomitantly, the global affinity in the presence of Ca²⁺ is reduced.</p