SUPPLEMENTARY FIGURE S1. 4.1R80 interaction with membrane proteins is
regulated by Ca2+ through CaM binding to FERM domain of 4.1R 80 (R30).
Effect of Ca2+ on 4.1R80 binding to cytoplasmic domains of transmembrane proteins
CD44 [6], GPC [10], NHE1 [8] and membrane scaffolding protein p55 [10] through
CaM binding to FERM domain (R30) are shown. Note that 4.1R 80 binds to CaM in a
Ca2+-independent manner with the same affinity as shown in Supplementary Table S3
[19]. A: schematic representations of 4.1R 80 and CaM are shown. 4.1R80 consists of 4
domains, including a N-terminal FERM domain (R30) and a spectrin and actin binding
(SAB) domain. B: In human erythrocyte membranes, 4.1R80 binds to both GPC, a
single transmembrane glycoprotein, and p55 through R30; it also forms a ternary
complex with spectrin and actin through the SAB domain. When CaM binds to R30
saturated with Ca2+, Ca2+/CaM weakens the binding affinities within the two ternary
protein complexes, 4.1R80/GPC/p55 and 4.180/spectrin/actin. The membrane skeletal
network becomes relaxed (i.e. it interacts more loosely with transmembrane proteins)
and the membrane stability is decreased [3,17]. C: upon activation of NHE1,
intracellular Ca2+ concentration increases. Upon Ca2+/CaM-dependent dissociation of
4.1R80 from NHE1, PIP2 binds to NHE1 and enhances NHE1 activity. (D) in epithelial
cells, 4.1R80 regulates CD44-hyalulonan interaction through ankyrin-CD44 interaction
[6].
A
B
C
D
SUPPLEMENTARY FIGURE S2. NMR structural changes in apo-CaM and
Ca2+/CaM upon interaction with pep11. Changes in NMR (1H-15N HSQC) spectral
profiles for specific amino acids in 15N-labelled apo-CaM (A) and Ca2+/CaM (B) upon
pep11 binding are shown. Spectra for the pep11/apo-CaM and pep11/Ca2+/CaM
complexes were measured at a 1:1.5 ratio. All samples were adjusted at pH 6.8 and
kept at 25°C during the experiments. Spectra for the pep11/apo-CaM and
pep11/Ca2+/CaM complexes were measured at a 1:1.5 ratio. All samples were adjusted
at pH 6.8 and kept at 25°C during the experiments.
A
B
- 105
+Ca2+
G33
G40
without pep11
G113
T29
G98
G25
G61
V55
T62
G132
G96
G23
with pep11
T44
G134
T26
D58
D95
T5
- 110
S17
D22
M145
T117
T110
T28
R106
N53
F65
Q135
T70
F19
- 115
S81 E127
M109
M72
R74 V91
15
E83
- 120
L18
L105
D118 R86
A102
D78
A73
S101 F68
K115
K21
L116
F141
- 125
A147
V136
I100
K94
I27
K148
D64
N137
I130
- 130
blue; residues in the N-lobe
A57
green; residues in the C-lobe
-
-
-
-
-
10
9
8
7
1
2
- 135
11
H (ppm)
N (ppm)
A128
F16
SUPPLEMENTARY FIGURE S3. Predicted hydrophobic residues in the C-lobe
of CaM represented as a 3D structure. The residues in pep11 involved in interaction
with CaM are represented as a ball-stick model in blue; the residues involved in Ca2+
independent interaction are represented as a sphere: L116 residue in yellow, A128
residue in brown, F141 residue in blue, M145 residue in green and A147 residue in
magenta. Location of the amino acid residue in CaM involved in CaM binding to
pep11, N137 in the -sheet when complexed with Ca2+ (PDB Accession ID: 1CFL for
apo-CaM and 1CLL for Ca2+/CaM), is also shown.
N137
138
N
y Figure S4.
l.
apo-CaM
A128
F141
A147
M145
L116
137
N
N138
Ca
A128
F141
Ca2+/CaM
M145
L116
3
A147
Kd for the interactions of CaM with R30 (Table S1) and pep11 (Table S2) in the
absence (EGTA) or presence (Ca2+) of Ca2+ from one of our previous reports [19] are
shown. Each analyte (used from 50nM to 2 µM) was incubated with the listed ligands
immobilized on aminosilane cuvettes. Interactions of R30 or pep11 with CaM in the
presence or absence of Ca2+ were examined using the IAsys ® resonant mirror detection
system as previously described [19,20]. Dissociation constants at equilibrium (termed
“Kd”) were calculated using the equation: Kd= kd ka-1, where ka is the association rate
constant and kd the dissociation rate constant. Kd values were obtained from the means
of 3-5 measurements for ka and kd.
From the binding curves obtained by the resonant
mirror detection method, Kd were determined using the software package FAST-Fit ®.
From the binding curves obtained by the resonant mirror detection method, Kd were
determined using the software package FAST-Fit ®. ka, kd and Kd were calculated from
three independent and representative experiments (mean±S.D.). ΔG°=RTlnKd、R=8.314
JK-1 mol-1, T / K.
4
Previously published results [6,8,10] about the effect of Ca2+/CaM on the affinity (Kd)
of R30 binding to the cytoplasmic domain of transmembrane proteins CD44 [6], GPC
[10] and NHE1 [8] and to membrane-associated protein p55 [10] are shown. R30 was
complexed or not with CaM then incubated at various concentrations (50 nM to 1 µM)
with the cytoplasmic domain of various transmembrane proteins immobilized on an
aminosilane cuvette in the absence (EGTA) or presence (Ca2+) of Ca2+ as described in
the Experimental section. Interactions of pep11 with CaM in the presence and absence
of Ca2+ and CaM were examined using the IAsys ® resonant mirror detection system
described as previously reported [19,20]. Dissociation constants at equilibrium (termed
“Kd”) were calculated using the equation: Kd= kd ka-1, where ka is the association rate
constant and kd the dissociation rate constant. Kd values were obtained from the means
of 3-5 measurements for ka and kd.
From the binding curves obtained by the resonant
mirror detection method, Kd were determined using the software package FAST-Fit ®.
ka, kd and Kd were calculated from three independent and representative experiments
(mean±S.D.). ΔG°=RTlnKd、R=8.314 JK-1 mol-1, T/K. Binding assays were performed
at 25°C.
5
Previously published results related to the Kd of pep11/CaM complex binding to pep9
are shown [19]. pep11/CaM complex (analyte used at 50nM to 2 µM) was incubated
with pep9 (ligands) immobilized on aminosilane cuvettes. Interactions of pep11/CaM
complex with pep9 in the presence and absence of Ca2+ were examined using the
IAsys® resonant mirror detection system as previously described [19]. Dissociation
constants at equilibrium (termed “Kd”) were calculated using equation: Kd = kd / ka,
where ka is the association rate constant and kd the dissociation rate constant. Kd values
were obtained from the means of 3-5 measurements for ka and kd. From the binding
curves obtained by the resonant mirror detection method, Kd were determined using the
software package FAST-Fit®. ka, kd and Kd were calculated from three independent and
representative experiments (mean±S.D.). ΔG°=RTlnKd 、 R=8.314 JK-1 mol-1, T /K.
Binding assays were performed at 25°C.
6