Kinetic characterization of apoptotic Ras signalling through Nore1-MST1
complex formation
Agne Koturenkiene, Cihan Makbul, Christian Herrmann and Diana ConstantinescuAruxandei
Supplementary material
Proteins expression and purification
All proteins were expressed in Escherichia coli (E.coli).
H-Ras (residues 1-189) was expressed and purified as described elsewhere (Herrmann et al.,
1996).
Murine Nore1A RBD (residues 199-358) and Nore1A RBD-SARAH (residues 199-413) were
expressed and purified similarly to Nore1A SARAH domain described previously (Makbul et
al., 2013). Briefly, the corresponding DNA fragments were cloned in pGEX-4T vectors (GE
Healthcare, Freiburg, Germany) and the resulting recombinant GST fusion proteins were
synthesized in E. coli strain BL21 DE3 according to the manufacturer’s recommendations.
The cells were resuspended in lysis buffer (50 mM Tris, 500 mM NaCl, 4 mM DTE, 1 mM
PMSF, 0.1% Triton X-100, pH 7.4) and lysed by ultrasonification for 10 minutes at 4ºC. After
centrifugation, the supernatant was loaded on a Glutathion Sepharose 4 Fast Flow column
(GE Healthcare, Freiburg, Germany) pre-equilibrated in lysis buffer and the unbound proteins
were washed extensively with buffer A (50 mM Tris, 500 mM NaCl, 2 mM DTE, pH 7.4) and
buffer B (50 mM Tris, 100 mM NaCl, 2 mM DTE, pH 7.4). After cleavage with thrombin, the
cleaved protein fragment was eluted from the column, concentrated by ultrafiltration and
further purified by chromatography, on a Superdex 200 HR 10/30 gel filtration column (GE
Healthcare, Freiburg, Germany) in buffer C (50 mM Tris, 100 or 200 mM NaCl, 2 mM DTE,
pH 7.4). Nore1A RBD-SARAH (residues 199-413) needed the presence of minimum 200 mM
NaCl in the buffers during the entire purification.
Human MST1 Inhibitory-SARAH (residues 330-487) was synthesized and purified as
previously described (Constantinescu Aruxandei et al., 2011).
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A modified version of vector pProEX-HTb (pProEX-HTb-eCFP-N) was used for MST1
Inhibitory-SARAH fused to eCFP at the N-terminus. The pProEX-HTb vector contained a Nterminal hexa-Histidine tag (His-tag), a spacer region of seven amino acids and a TEV
protease cleavage site. The protein was purified on a Nickel-NTA Super flow column
(Qiagen, Hilden, Germany). The filtered sample in lysis buffer (50 mM Tris, 500 mM NaCl,
20 mM Imidazole, 1 mM PMSF, 0.1% Triton X-100, pH 7.4) was loaded on the equilibrated
column and the unbound proteins were washed with 50 mM Tris, 100 mM NaCl, 20 mM
imidazole, pH 7.4. The target protein was eluted by a linear gradient from 20 mM to 500 mM
imidazole.
A
B
C
D
E
Figure S1. Analytical size exclusion chromatography and SDS-PAGE of H-Ras, Nore1A RBD/RBDSARAH and Nore1A RBD/RBD-SARAH – Ras complexes: H-Ras does not disrupt the Nore1A RBDSARAH homodimer.
(A) Elution profiles of H-Ras.GppNHp, Nore1A RBD (residues 199-358) and a 1:1 mixture of these two
proteins; (B) Position of the eluted proteins (red circles) from (A) on the calibration curve; (C) Elution profiles
of H-Ras.GppNHp, Nore1A RBD-SARAH (residues 199-413) and a 1:1 mixture of these two proteins; (D)
Position of the eluted proteins (red circles) from (C) on the calibration curve; (E) SDS-PAGE from the eluted
fractions of (1) Nore1A RBD-SARAH, (2) H-Ras.GppNHp and (3) the shaded area of the 1:1 mixture of these
two proteins in (C).
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A
B
Figure S2. Analytical size exclusion chromatography of Nore1A RBD-SARAH (green), MST1 InhibitorySARAH (cyan) and a 1:1 mixture of these two proteins (black).
(A) Elution profiles of Nore1A RBD-SARAH (green), MST1 Inhibitory-SARAH (cyan) and a 1:1 mixture of
these two proteins (black). The apparent molecular weights above were calculated from the calibration curve (B)
which also shows the position of the eluted proteins from (A).
A
B
C
D
E
F
Figure S3. Analytical size exclusion chromatography of Nore1A RBD-SARAH, MST1 Inhibitory-SARAH
fused to eCFP, H-Ras, Nore1A RBD-SARAH – MST1 Inhibitory-SARAH and Nore1A RBD-SARAH –
MST1 Inhibitory-SARAH – H-Ras complexes: Nore1A RBD-SARAH, MST1 Inhibitory-SARAH and HRas form a ternary complex.
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(A) Elution profiles of Nore1A RBD-SARAH, MST1 Inhibitory-SARAH fused to eCFP and a 1:1 mixture of
these two proteins; (B) Position of the eluted proteins from (A) on the calibration curve; (C) SDS-PAGE from
the eluted fractions of (1) MST1 Inhibitory-SARAH, (2) Nore1A RBD-SARAH, (2) and (3) the shaded area of
the 1:1 mixture of these two proteins in (A); (D) Elution profiles of H-Ras.GppNHp, Nore1A RBD-SARAH,
MST1 Inhibitory-SARAH and a mixture of these three proteins; (E) Position of the eluted proteins from (D) on
the calibration curve; (E) SDS-PAGE from the eluted fractions of (1) H-Ras.GppNHp, (2) Nore1A RBDSARAH, (3) MST1 Inhibitory-SARAH and (4-7) the shaded area (from left to right) of the mixture of these three
proteins in (D).
Figure S4. Nore1A RBD-SARAH inhibition of mGppNHp dissociation from H-Ras.
The nucleotide dissociation was measured as exponential fluorescence decrease at λem 450 nm (λex 360 nm). The
measurement conditions were: 37°C, 50 mM Tris, 5 mM MgCl 2, 2 mM DTE, 200 mM NaCl, pH 7.4. The GDI
assays were performed with 1000x excess of unlabeled versus fluorescently labeled GppNHp (mGppNHp) to
prevent backward association of mGppNHp to H-Ras. The dissociation kinetics can be analysed as monoexponential decay under these conditions. The dissociation rate constants of fluorescently labelled mGppNHp,
kobs, in the presence of various concentrations of Nore1A RBD-SARAH have been measured and plotted as
a function of Nore1A RBD-SARAH concentration (Figure 1 in the main text).
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Figure S5. Nore1A RBD-SARAH association with H-Ras.mGppNHp measured by stopped-flow.
Rapid mixing of H-Ras bound to fluorescently-labelled unhydrolysable GTP analogue mGppNHp with
increasing concentration of Nore1A RBD-SARAH produces observable fluorescence change. Under pseudo
first-order conditions with Nore1A RBD-SARAH concentration being at least 10-fold higher than H-Ras, the
observed fluorescence change in time can be fitted with a mono-exponential curve yielding an observed rate
constant kobs. The association was measured as exponential fluorescence decrease with λex = 366 nm, cut-off
filter 420 nm. The experiment was performed at 10°C, in 50 mM Tris (pH 7.4), 5 mM MgCl 2, 2 mM DTE, 200
mM NaCl.
Figure S6. Kinetics of Nore1A RBD dissociation from H-Ras.mGppNHp measured by stopped-flow.
The dissociation was measured as exponential fluorescence increase with λex 366 nm, cut-off filter 420 nm. The
experiment was performed at 10°C in 50 mM Tris buffer (pH 7.4), 5 mM MgCl 2, 2 mM DTE, 200 mM NaCl.
The fluorescence change (increase) due to H - Ras.mGppNHp dissociation from Nore1A RBD was fitted with
mono-exponential equation, yielding dissociation rate constant koff = 0.3 s-1.
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A
B
Figure S7. DSC data of Nore1A RBD and RBD-SARAH.
(A) Thermal unfolding profiles of 40 μM Nore1A RBD (green line) and of 80 μM Nore1A RBD-SARAH
(blue line). The solid lines represent the first scan and the dashed line is the second scan. The buffer was 50 mM
Tris, 200 mM NaCl, pH 7.4 and the scan rate was 40 deg min-1. The thermograms show exothermic peaks at high
temperature, characteristic to aggregation. (B) SDS-Page of Nore1A RBD (lanes 1 and 2) and of Nore1A RBDSARAH (lanes 3 and 4) before (lanes 1 and 3) and after (lanes 2 and 4) the first DSC scan. The proteins
precipitated during the DSC scan, as no soluble band can be observed on the SDS-PAGE (lanes 2 and 4).
Table S1. Kinetic parameters of Nore1A binding to H-Ras.mGppNHp.
Linear range
Nore1A
NaCl
kon (µM-1 s-1)
Saturation
koff (s-1)
KD (µM)
k2 (s-1)
K1 (µM)
(µM-1 s-1)
(mM)
RBD
RBD-
kon = k2/K1
0
10.57
0.12
0.012
204
10
20.4
200
2.1
0.3
0.14
243
86
2.8
200
3.6
0.15
0.042
201
51
3.9
SARAH
The data were obtained using stopped-flow at 10ºC with λex 366 nm and 420 nm cut-off filter.
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References
Constantinescu Aruxandei, D., Makbul, C., Koturenkiene, A., Lüdemann, M.B., Herrmann, C.
(2011). Dimerization-induced folding of MST1 SARAH and the influence of the
intrinsically unstructured inhibitory domain: Low thermodynamic stability of
monomer. Biochemistry 50, 10990-11000.
Herrmann, C., Horn, G., Spaargaren, M., Wittinghofer, A. (1996). Differential interaction of
the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative
effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. J Biol
Chem 271, 6794-800.
Makbul, C., Constantinescu Aruxandei, D., Hofmann, E., Schwarz, D., Wolf, E., Herrmann,
C. (2013). Structural and thermodynamic characterization of Nore1-SARAH: a small,
helical module important in signal transduction networks. Biochemistry 52, 1045-54.
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