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Kim et al.
Supplementary Information
Supplementary Figure 1 a, Superposition of the isolated Ca2+/CaM-CaV1.2 IQ domain complexes
from 2BE6 (Van Petegem et al, 2005) (conformation A, yellow) and 2F3Y (Fallon et al, 2005) (white)
with the corresponding complex from Ca2+/CaM-CaV1.2 PreIQ-IQ domain structure from Figure 1a.
b, Superposition of the Ca2+/CaM-CaV1.2 PreIQ-IQ domain complexes presented here (colored as in
Figure 1a) and from Fallon et al. (Fallon et al, 2009)(white). CaM N-lobe and C-lobe, PreIQ-IQ
domain N-terminus, PreIQ, and IQ domains are indicated.
Supplementary Figure 2 Superdex 200 size exclusion chromatography of a, Ca2+/CaM-CaV1.2
PreIQ-IQ complex and b, Ca2+/CaM-CaV1.2 C-IQ complex. Elution volumes of molecular weight
standards are indicated.
Supplementary Figure 3 The 1:1 Ca2+/CaM-CaV1.2 PreIQ-IQ complex contains Ca2+/CaMIQ.
a, Reapplication of the 1:1 Ca2+/CaM-CaV1.2 PreIQ-IQ complex (Figure 4a, Peak 1) onto
phenylsepharose. No further CaM was eluted upon addition of 10 mM EGTA. b, Phenylsepharose
chromatography of the isolated Ca2+/CaM-CaV1.2 IQ complex. No CaM was detected in the 10 mM
EGTA elution. Absorbance and conductivity are shown in blue and green, respectively.
Supplementary Figure 4 Comparison of the circular dichroism spectra of a, N-lobe in EGTA
(open circles), Ca2+/N-lobe (black circles), and Apo/N-lobe12 (green circles), and b, C-lobe in EGTA
(open circles), Ca2+/C-lobe (black circles), and Apo/C-lobe34 (blue circles). Spectra were recorded at
20 M protein and 15°C. Spectra for N-lobe and C-lobe were collected in 5 mM KCl, 10 mM EGTA,
10 mM K+-HEPES, pH 7.4. Spectra for Ca2+/N-lobe, Apo/N-lobe12, Ca2+/C-lobe, and Apo/C-lobe34
were collected in 5 mM KCl, 1 mM CaCl2, 10 mM K+-HEPES, pH 7.4.
Supplementary Figure 5 Relative current amplitude increase from the first to the fortieth pulses for
CaV1.2 with mutations in the Ca2+/CaMAC-PreIQ binding sites and the coiled-coil interface. To unmask
CDF, all mutants were measured on the background of Ile1624Ala mutation. Results of unpaired t test
are indicated as follows: N.S., not significant, P > 0.05; ‘*’, 0.01 < P < 0.05; ‘**’, 0.001 < P < 0.01;
‘***’, P < 0.001. Error bars indicate S.D.
Supplementary figure 6 Comparison of CaV1.2 and NaV1 EF-hand regions. a, Sequence alignments
of human NaV1.2 (2KAV) (Miloushev et al, 2009), human NaV1.5 (2KBI) (Chagot et al, 2009), and
human CaV1.2 (Cavα1c77, Accession number CAA84346) EF-hand regions. Secondary structures
indicated above the alignment are based on the NaV1.2 NMR structure (2KAV) (Miloushev et al, 2009).
Identical residues and type-conserved residues are shaded in orange and light orange, respectively.
Orange and cyan filled circles below the alignments correspond the residues in the EF-hand domain
hydrophobic core displayed in ‘c’. EF hand positions are indicated in grey along with the most
common amino acid at each position (Gifford et al, 2007). b, Model of the CaV1.2 EF-hand (orange)
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Kim et al.
based on NaV1.2 (2KAV) (white). CaV1.2 A-region is highlighted in cyan. Helix I, Helix IV, and Nand C-termini are indicated. c, Close-up view of the hydrophobic core sidechains of the CaV1.2 EFhand homology model (orange and cyan) and NaV1.2 (2KAV) (white). CaV1.2 residue numbers are in
orange and cyan. NaV1.2 residue numbers are in black. Model suggests that CaV1.2 L1565 interacts
extensively with surrounding hydrophobic residues. Color-code is as in ‘b’.
Supplementary References
Chagot B, Potet F, Balser JR, Chazin WJ (2009) Solution NMR structure of the C-terminal EF-hand
domain of human cardiac sodium channel NaV1.5. J Biol Chem 284(10): 6436-6445
Fallon JL, Baker MR, Xiong L, Loy RE, Yang G, Dirksen RT, Hamilton SL, Quiocho FA (2009)
Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca2+
calmodulins. Proc Natl Acad Sci U S A 106(13): 5135-5140
Fallon JL, Halling DB, Hamilton SL, Quiocho FA (2005) Structure of calmodulin bound to the
hydrophobic IQ domain of the cardiac Ca(v)1.2 calcium channel. Structure 13(12): 1881-1886
Gifford JL, Walsh MP, Vogel HJ (2007) Structures and metal-ion-binding properties of the Ca2+binding helix-loop-helix EF-hand motifs. Biochem J 405(2): 199-221
Miloushev VZ, Levine JA, Arbing MA, Hunt JF, Pitt GS, Palmer AG, 3rd (2009) Solution structure of
the NaV1.2 C-terminal EF-hand domain. J Biol Chem 284(10): 6446-6454
Van Petegem F, Chatelain FC, Minor DL, Jr. (2005) Insights into voltage-gated calcium channel
regulation from the structure of the CaV1.2 IQ domain-Ca2+/calmodulin complex. Nat Struct Mol
Biol 12(12): 1108-1115
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