1. Volume 24, Issue 10, Pages 1742-1754 (October 2016)
Molecular Insights into the Mechanism of Calmodulin Inhibition of the EAG1 Potassium Channel 
Maria João Marques-Carvalho, Johannes Oppermann, Eva Muñoz, Andreia S. Fernandes, Guillaume Gabant, Martine Cadene, Stefan H. Heinemann, Roland Schönherr, João Henrique Morais-Cabral 
Structure 
Volume 24, Issue 10, Pages (October 2016)
DOI: /j.str
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2. Figure 1 Characterization of the Interaction of CaM with BDC2
(A) ITC titration of 60 μM CaM into 6 μM MBP-BDC2S.
(B) Titration of 60 μM CaM into 6 μM MBP-BDC2L.
(C) Titration of 180 μM N-lobe CaM into 19 μM MBP-BDC2L.
(D) Titration of 100 μM C-lobe CaM into 10 μM MBP-BDC2L.
Upper graphs show heat-power changes and lower graphs show integrated heat values fitted with a model curve. Cartoons represent components in each titration, with CaM lobes (N and C, as labeled) shown as circles, full-length CaM represented by linked circles, and Ca2+ bound in lobes shown as closed dots; lobes without Ca2+ have no dots. Experiments performed in saturating Ca2+ concentrations (5 mM).
Structure  , DOI: ( /j.str )
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3. Figure 2 Structure of Calmodulin in Complex with BDC2
(A) Full view of Ca2+-CaM-BDC2 structure with CaM in red and BDC2 in cyan. Calcium ions are represented by black spheres.
(B) Zoom over the C lobe (shown as surface) and BDC2 channel fragment (shown as sticks).
(C) View of the crystal lattice with neighboring CaM molecules in lilac.
(D) View of residues in hydrophobic cluster. Only side chains are shown, cyan for BDC2 and red for CaM. van der Waals contacts (≤4 Å) are shown as dashed lines. Ca2+ is shown as spheres.
(E) View of residues in elbow of BDC2, with potential hydrogen bond network (donor and acceptor at ≤3.5 Å) shown as dashed lines.
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4. Figure 3 Characterization of BDC2 Mutants
(A) Dissociation constant ratios for wild-type (wt) CaM-BDC2 complex relative to mutants. Errors are SD.
(B) Time course of normalized current inhibition after elevation of intracellular Ca2+ by extracellular exposure to 1 μM ionomycin. Wild-type and mutant hEAG1 channels were expressed in Xenopus oocytes and currents were recorded in two-electrode voltage clamp. Averaged currents during repetitive test pulses (40 mV) were normalized to current amplitudes before exposure to ionomycin. Numbers of independent experiments are in parentheses.
(C) Quantification of steady-state inhibition seen in (B).
Errors bars denote SEM. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant. Equivalent mutations are shown as bars of the same color in (A) and (C).
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5. Figure 4 Characterization of the Interaction of Calmodulin with BDN
(A) ITC titration of 60 μM CaM into 6 μM MBP-BDNS.
(B) Titration of 60 μM CaM into 6 μM MBP-BDNL.
(C) Titration of 253 μM N lobe into 23 μM MBP-BDNL.
(D) Titration of 258 μM C lobe into 23 μM MBP-BDNL.
Organization of panels and symbols as in Figure 1. Experiments performed in saturating Ca2+ concentrations (5 mM).
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6. Figure 5
BDC1 Mutations Affect Interaction between PAS and CNBh Domains
(A) Interface of complex formed by mEAG1 PAS (cyan and gray) and CNBhD-BDC1 (blue and gray) (Haitin et al., 2013). C terminus of CNBhD-BDC1 corresponds to either residue 717, 718, or 720 depending of which of the four complexes present in the crystal structure (PDB: 4LLO) is analyzed. Intrinsic ligand residues in CNBh domain are shown; BDC1 region and residues in PAS domain that interact with BDC1 are depicted as sticks.
(B) Fluorescence anisotropy of fluorescein-labeled PAS domain titrated with wild-type CNBhD-BDC1 (squares) and with same protein mutated in the BDC1 region: R702N/R704N/R708N/K709N (circles) and I705A/V706A/F707A (crosses). Errors are SD.
Structure  , DOI: ( /j.str )
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7. Figure 6
Characterization of the Interaction of Calmodulin with CNBhD-BDC1-BDC2
(A) ITC titration of 187 μM CaM into 17 μM CNBhD-BDC1-BDC2, fitted with model considering two sets of independent sites on the channel fragment.
(B) Titration of 140 μM N lobe into 14 μM CNBhD-BDC1-BDC2.
(C) Titration of 100 μM C lobe into 10 μM CNBhD-BDC1-BDC2.
(D) Titration of 100 μM CaM-EF12 into 100 μM CNBhD-BDC1-BDC2.
(E) Titration of 60 μM CaM into 6 μM MBP-BDC1-BDC2, fitted as in (A).
(F) Titration of 70 μM CaM into 6 μM MBP-linkerBDC2, fitted as in (A).
(G) Titration of 60 μM CaM into 6 μM CNBhD-BDC1-BDC2 BDC1 mutant R702N/R704N/R708N/K709N, fitted as in (A).
Arrows indicate steps in binding isotherms. Organization of panels and symbols as in Figure 1; mutated Ca2+ binding sites are shown as small crosses, while large cross indicates mutations in BDC1. Experiments performed in saturating Ca2+ concentrations (5 mM).
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8. Figure 7 Modeling of Calmodulin Binding to CNBh-BDC1-BDC2
(A) CaM independent-site binding model. k represents site-specific binding constants for each lobe (N or C). Cartoons as in Figure 1.
(B) Titration of 187 μM CaM into 17 μM CNBhD-BDC1-BDC2 (same data as in Figure 5A) fitted with model shown in (A), together with respective species distribution plot.
(C) Titration of 100 μM C lobe into a mixture of 7 μM CNBhD-BDC1-BDC2 and 35 μM N lobe and species distribution plot. All experiments were performed in saturating Ca2+ concentrations (5 mM).
Structure  , DOI: ( /j.str )
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