4 AMPK Activity (Fold Activation) 3 2 0.1 1 10 100 1000 ... · curve of AMPK activity (fold...

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1 Supplementary Figure 1: Effect of high ATP concentration on AMP activation of AMPK α1. AMP dose-response (0 – 1 mM) at near-physiological (2 mM) ATP concentration; results from six independent experiments were plotted as a dose-response curve of AMPK activity (fold activation ± SEM) vs [AMP] (µM). The values for EC 50 , fold stimulation and Hill coefficients were calculated based on fitting the data to the equation: Activity = Basal + (((Fold stimulation x Basal) – Basal x [C2] h )/ ((EC 50 ) h + [C2] h )). 0 1 2 3 4 5 6 0.1 1 10 100 1000 10000 AMPK Activity (Fold Activation) [AMP] (μM)

Transcript of 4 AMPK Activity (Fold Activation) 3 2 0.1 1 10 100 1000 ... · curve of AMPK activity (fold...

Page 1: 4 AMPK Activity (Fold Activation) 3 2 0.1 1 10 100 1000 ... · curve of AMPK activity (fold activation ± SEM) vs [AMP] (µM). The values for EC ... C2 AMP EC 50 (unit) 50.3 nM 158.1

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Supplementary Figure 1: Effect of high ATP concentration on AMP activation of

AMPK α1. AMP dose-response (0 – 1 mM) at near-physiological (2 mM) ATP

concentration; results from six independent experiments were plotted as a dose-response

curve of AMPK activity (fold activation ± SEM) vs [AMP] (µM). The values for EC50,

fold stimulation and Hill coefficients were calculated based on fitting the data to the

equation: Activity = Basal + (((Fold stimulation x Basal) – Basal x [C2]h)/ ((EC50)h +

[C2]h)).

0

1

2

3

4

5

6

0.1 1 10 100 1000 10000

AM

PK A

ctiv

ity(F

old

Act

ivat

ion)

[AMP] (µM)

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Supplementary Figure 2: Western blotting of AMPK protein. (a) Western blot of

AMPK "1 and "2 showing the levels of AMPK "-pT172, total "-AMPK, #-pS108 and

total #1-AMPK following treatment with $-phosphatase or CaMKK2. (b) Expression of

AMPK %1 WT and its three respective mutants (%T89E, %H151E and %H298E). Levels

were detected using immunoblotting with FLAG-antibody for AMPK "-subunit and HA-

tag antibody for %-subunit. Images have been cropped to improve clarity of the figure.

Original blots were displayed in Appendix Ai-iii.

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IB: FLAG!!

HA!

HA-"1!

75!!!

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100!!

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37!

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Supplementary Figure 3: αRIM interactions with the γ-subunit. (a) Stereo omit

electron density map (2Fo-Fc) for the C2 molecules, contoured at 2.0 σ (gray mesh). (b)

Differences between C2-bound (α = green; γ = magenta) and AMP-bound (α = dark

gray; γ = white) heterotrimers. Electron density for the αRIM2 was only visible in the

AMP-bound heterotrimer. (c) Comparison of the αRIM2 motif of AMPK isoforms;

superposition of the αRIM2 motif from α1 (cyan; PDB 4RER) and α2 (red; PDB 4CFE),

with γ1 from a α2β1γ1 (PDB 4CFE) shown in surface representation.

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Supplementary Figure 4: C2 and AMP binding to the γ-subunit causes distinct

conformational changes. The γ-subunit (magenta) is shown in cartoon representation,

and C2 (yellow), AMP (grey) and selected side-chains (α = green; γ = grey) in stick

representation. (a) Two conformations of γHis298. The conformational differences are

necessary to avoid a clash with AMP (white γHis298) or C2 (magenta γHis298). (b) Two

conformations of γArg70. Binding of C2 disrupts the hydrogen bond (black dashed line)

between γArg70 and αGlu368 (AMP-bound; white and green, respectively). The close

contact with C2 moves γArg70 (magenta) ~ 4.0 Å, clashing with αGlu368 (C2-bound;

not modeled) in this new conformation and disengaging the αRIM.

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Supplementary Table 1: Values for EC50, Vmax and Hill coefficients for C2 (Fig. 1d)

and AMP (Supplementary Fig. 1) at near-physiological ATP (2 mM), calculated based

on fitting the data to the equation: Activity = Basal + (((Fold stimulation x Basal) – Basal

x [C2]h)/ ((EC50)h + [C2]h)).

ATP (2mM) C2 AMP

EC50 (unit) 50.3 nM 158.1 µM

Fold stimulation 6.7 ± 0.2 5.4 ± 0.6

Hill coefficient 2.3 ± 0.4 1.0 ± 0.2

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Supplementary Note 1: Western blot of AMPK "1 showing the levels of AMPK "-

pT172, total "-AMPK, #-pS108 and total #1-AMPK following treatment with $-

phosphatase or CaMKK2 (Supplementary Fig. 2a). All blots displayed are uncropped

scans from Odyssey phosphoimager.

250 150 100 75

!AMPK-IR800

!pT172-IR680

"-phosphatase + - CaMKK2 - +

GST-!1AMPK MW

250 150 100 75

GST-!1AMPK MW

#1AMPK-IR800

#pS108-IR680

"-phosphatase + - CaMKK2 - +

50

37

25 50

37

25

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Supplementary Note 2: Western blot of AMPK "2 showing the levels of AMPK "-

pT172, total "-AMPK, #-pS108 and total #1-AMPK following treatment with $-

phosphatase or CaMKK2 (Supplementary Fig. 2a). All blots displayed are uncropped

scans from Odyssey phosphoimager.

150

100

75

MW GST-!2AMPK

"-phosphatase + - CaMKK2 - +

150

100

75

!AMPK-IR800

!pT172-IR680

#1AMPK-IR800

#pS108-IR680

100 75

50

37

25

20

100 75

50

37

25

"-phosphatase + - CaMKK2 - +

GST-!2AMPK MW

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Supplementary Note 3: Western blot of the expression of AMPK %1 WT and its three

respective mutants (%T89E, %H151E and %H298E; Supplementary Fig. 2b), with

appropriate control (GST"-AMPK) included. Levels were detected using immunoblotting

with FLAG-antibody for AMPK "-subunit and HA-tag antibody for %-subunit. All blots

displayed are uncropped scans from the Odyssey phosphoimager.

AMPK HA!- IR800

50

37

50 50

AMPK "FLAG- IR680

100

75

50

37

wt T89E H151E H298E GST"-AMPK

Merged

100

75

50

37

HA-!1

MW