Supporting InformationLaursen et al. 10.1073/pnas.1422997112

A

B

C

Cardiotonicsteroid

Subs�tuents at the steroid coreClass of CTS

1β 3β 5β 11α 12β 14β 19β

Bufalin -OH -OH bufadienolide

Digitoxigenin -OH -OH cardenolide

Digoxin tri-D-digitoxose -OH -OH cardenolide

Ouabain -OH L-rhamnose -OH -OH -OH -OH cardenolide

Ouabagenin -OH -OH -OH -OH -OH -OH cardenolide

Fig. S1. Structure of CTSs. (A) The structural core of cardenolides. (B) The structural core of bufadienolides. (C) Overview of CTSs investigated in this report anda table of their substituents at the steroid core.

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BA

C D

c

a

c

b

c

b

c

a

Fig. S2. Crystal packing. Crystals of the E2P–bufalin and the E2P–digoxin complexes were obtained in two different crystal forms with space group symmetriesP21 and P212121, respectively. Each crystal form displays a type I crystal packing, with layers of membranes stacked on top of each other (1). For both crystalforms, the asymmetric unit contains two oppositely oriented αβγ-complexes, which are highlighted in bright colors. The three cytoplasmic domains (the ac-tuator domain, the nucleotide-binding domain, and the phosphorylation domain), are depicted in yellow, red, and cobalt blue colors, respectively. The re-spective unit cells are indicated. (A and B) Crystal lattice of the E2P–bufalin complex viewed down axes a and b, respectively. (C and D) Crystal lattice of the E2P–digoxin complex viewed down axes a and b, respectively.

1. Michel H (1983) Crystallization of membrane proteins. Trends Biochem Sci 8(2):56–59.

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C

αM4

αM3

αM5

αM6

D

αM4

αM3

αM5

αM6

B

αM4

αM3

αM5

αM6

A

αM4

αM3

αM5

αM6

II

I

6.0σ level

4.0σ 0.3level σ level

5.0σ level

bufalin

5.0σ 0.3level σ level

αM4

αM3

αM5

αM6

loop αM7-8

F

αM4

αM3

αM5

αM6

G

6.5σ level

αM4

αM3

αM5

αM6

loop αM7-8

E

digoxin

Mg2+

Fig. S3. Verification of the position of the bufalin and digoxin molecules and cations in the binding sites. The α-subunit is represented by gray cartoon, bufalinand digoxin are represented by magenta and orange sticks, and potassium and magnesium are represented by purple and yellow spheres, respectively. Theinitial Fo-Fc maps obtained after rigid body refinement are depicted by cyan mesh. For the E2P–bufalin complex, the noncrystallographic symmetry-averagedFo-Fc map is depicted at contour levels (A) 6.0σ, (B) 5.0σ, (C) 4.0σ, and (D) 3.0σ. Based on the strong signal from the aromatic lactone, the position of the bufalinmolecule could be identified. Also, the location of the two potassium ions could be assigned in the residual electron density. For the E2P–digoxin complex theinitial Fo-Fc map is depicted at contour levels (E) 6.5σ, (F) 5.0σ, and (G) 3.0σ, revealing the location of the Mg2+ and the digoxin molecule. The two distaldigitoxose units only appear at lower signal-to-noise ratio. However, stereochemical restraints (i.e., bond lengths, angles, and stereo chemistry) confined theflexibility of the tridigitoxose moiety to the glycosidic bonds (assuming that all digitoxose units are in the more favorable chair conformation), which onlyallowed one orientation of the two distal digitoxose units to fit the observed electron density.

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T772E327

V325

A323

E779

D804

V322

V322

αM3

αM4

αM5

αM6

I

II

Fig. S4. Structural alignment of the Na+,K+-ATPase E2P–bufalin complex with the [K2]E2–MgFx complex (1) performed on αM7–10 reveals structural re-arrangements of the K+-binding site. The E2P–bufalin complex is depicted in blue cartoons, with bufalin and K+ ions represented by magenta sticks and purplespheres, respectively. The [K2]E2–MgFx complex is depicted in black cartoons, and its K+ ions are depicted by gray spheres. Residues lining the K+-binding sitesare shown in sticks. The alignment further reveals that the winding/unwinding of αM4 makes Val322 (αM4) move in and out of the site as part of the gatingmechanism, where the hydrophobic Val322 closes the cation-binding site to the extracellular side. In the wound-up form of αM4, Val322 turns ∼90° away fromthe site, but when K+ ions bind and drag αM4 toward the site, Val322 rotates and closes the site from the extracellular side.

1. Morth JP, et al. (2007) Crystal structure of the sodium-potassium pump. Nature 450(7172):1043–1049.

0.0 0.5 1.0 1.5 2.0 2.5 3.00

25

50

75

100

Resid

ual N

a,K-

ATPa

se (%

)

CTS (µM)

Fig. S5. Effect of ionic strength on the binding of bufalin (□ and ■) and digitoxigenin (● and ○) to Na+,K+-ATPase. Ionic strength was increased by additionof 200 mM NMDG (□ and ○). The obtained Kd values were 14 ± 11 (bufalin) and 26 ± 15 nM (digitoxigenin).

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0.00 0.05 0.10 0.15 0.20 0.256.0

6.5

7.0

7.5

8.0

bufalinouabain

control

Fluo

resc

ence

(a.u

.)time (s)

Fig. S6. Changes in eosin fluorescence induced by its binding to Na+,K+-ATPase. The Na+,K+-ATPase was preincubated with bufalin or ouabain in the nominalabsence of Mg2+ (20 mM 1,2-diaminocyclohexanetetraacetic acid) before mixing with eosin (details in Materials and Methods). Traces are lined up to time0 and shifted with respect to each other to ease the comparison of the rates of fluorescence change. The obtained rate constants for eosin binding were 42.57 ±0.27 (control), 42.63 ± 0.35 (ouabain), and 14.13 ± 0.22 s−1 (bufalin).

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Table S1. Data collection and refinement statistics

[Mg]E2P–digoxin [K2]E2P–bufalin [Rb2]E2P–bufalin

Data collectionX-ray source X06SA X06SA X06SAWavelength (Å) 1.0 1.0 0.8Space group P212121 P21 P21Unit cell parameters

a, b, c (Å) 118.2, 118.3, 494.1 65.9, 240.3, 152.7 65.5, 239.4, 151.7α, β, γ (o) 90.0, 90.0, 90.0 90.0, 102.3, 90.0 90.0, 102.3, 90.0

Resolution range† (Å)Upper limit along a*, b*, c* 5.2, 4.3, 3.9 (4.0–3.9)‡ 5.6, 4.1, 3.4 (3.5–3.4)‡ 6.0, 6.0, 6.0 (6.1–6.0)§

Lower limit 50.0 55.0 45.0Unique reflections† 44,534 (181) 31,754 (68) 22,381 (1062)Redundancy† 5.6 (4.8) 5.9 (5.7) 3.5 (3.5)Completeness (%)

Low-resolution cutoff§{ 99.4 (99.7) 99.3 (100.0) 99.4 (99.6)High-resolution cutoff†‡# 68.6 (3.8) 50.2 (1.4)

Rmeas (I) (%)†k 8.2 (98.0) 13.3 (86.6) 11.1 (65.2)I/σ(I)* 11.1 (1.8) 10.4 (2.2) 9.9 (2.1)CC-1/2 (all data)†** 99.8 (62.0) 99.9 (75.0) 99.8 (85.8)

Refinement statisticsResolution range† (Å) 50.0–4.0 (4.1–4.0) 50.0–3.4 (3.5–3.4)Reflections 43,077 31,546Rwork

† (%) 22.1 (30.1) 24.5 (41.8)Rfree

† (%) 25.3 (34.3) 28.8 (32.3)No. of atoms (a.u.)

Protein 20,680 20,646Ligands 443 235

Average B factors (Å2)Protein 198.5 125.5Ligands 186.6 104.9

rmsdsBonds (Å) 0.009 0.011Angles (o) 1.128 1.225

Ramachandran plot†† (%)Favored 94.51 94.23Allowed 5.26 5.46Outliers 0.23 0.31

†Values for the highest-resolution shell are given in parentheses.‡Elliptical data truncation.§Spherical data truncation.{For [Mg]E2P–digoxin, 50.0- to 5.2-Å resolution, with values for resolution shell 5.3–5.2 Å given in parentheses.For [K2]E2P–bufalin, 55.0- to 5.6-Å resolution, with values for resolution shell 5.7–5.6 Å given in parentheses.#For [Mg]E2P–digoxin, 50- to 3.9-Å resolution. For [K2]E2P–bufalin, 55.0- to 3.4-Å resolution.kRmeasðIÞ=

Ph

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffinh=ðn−1Þp

×Pnh

i jI∧h − Ih,i j=

Ph,i Ih,i .

**Pearson correlation coefficient between intensities from random half-datasets (1).††Values given by the MolProbity server (2).

1. Karplus PA, Diederichs K (2012) Linking crystallographic model and data quality. Science 336(6084):1030–1033.2. Davis IW, et al. (2007) MolProbity: All-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 35(web server issue):W375–W383.

Table S2. Kinetic parameters of CTS binding under steady-stateconditions of the Na+,K+-ATPase reaction

Bufalin Ouabain

k−1 (s−1) ∼0.0002 ∼0.0002k+1 (μM−1s−1) 0.137 0.06113Ki (μM) 50.5 292.5

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