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Structural insights into xenobiotic and inhibitor binding

to human Aldehyde Oxidase

Supplementary Information

Catarina Coelho1, Alessandro Foti2, Tobias Hartmann2, Teresa Santos-Silva1, Silke Leimkühler2 and Maria João Romão1

1UCIBIO@REQUIMTE – Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

2Institute of Biochemistry and Biology, Department of Molecular Enzymology, University of Potsdam, D-14476 Potsdam, Germany

Nature Chemical Biology: doi:10.1038/nchembio.1895

PDB ID

Xanthine Oxidase (XO)

1FIQ; 3EUB; 3AX7; 3NVY; 3NVW; 3ETR; 3NVZ;

3NRZ; 3B9J; 3NVV; 3NS1; 2CKJ; 1SB3; 1RM6;

2E3T; 3SR6; 3AMZ; 3AX9; 2E1Q.

Xanthine Dehydrogenase (XDH)

1FO4; 3UNI; 3UNA; 3AM9; 3UNC; 1WYG;

3BDJ; 1N5X; 1VDV; 1V97; 3AN1; 2W54.

Supplementary Table1: PDB identification of the 31 XO and XDH structures analyzed.


hAOX1 hAOX1-Pht-Thi

Data collection

Space group P42212

Cell dimensions

a = b, c (Å) 148.90, 133.30 148.70, 132.80

Resolution (Å) 74.46-2.60 (2.69-

2.60)

49.58-2.70 (2.81-

2.70)

Rmerge 0.18 (2.73) 0.08 (1.08)

I / s(I) 11.20 (1.50) 22.20 (1.60)

Completeness (%) 100.00 (100.00) 99.80 (98.70)

Redundancy 16.10 (15.80) 12.00 (6.50)

Refinement

Resolution (Å) 2.60 2.70

No. reflections 46663 (4476) 41472 (4556)

Rwork / Rfree 18.8 / 23.3 19.7 / 24.4

No. atoms

Protein 10077 10089

Ligand/ion 14 74

Water 43 60

B-factors

Protein 47.39 53.14

Ligand/ion 50.42 85.58

Water 56.81 58.89

R.m.s. deviations

Bond lengths (Å) 0.008 0.007

Bond angles () 1.186 1.164

*Values in parentheses are for highest-resolution shell

Supplementary Table2: Data collection and refinement statistics


I440

Q430

Q1235

T1230

FeSII

FAD Variable loop

hAOX1:430QAQRQENALAI440

mAOX3:430QAPRQQNAFAT440

bXO/bXDH:423QASRREDDIAK433bXO/bXDH:423**S*::::I*K433

Supplementary Figure1: Superposition of hAOX1 (4uhw, in blue), mAOX3 (3zyv, in gray), bXO (1fiq, in pink) and bXDH (1fo4, in orange) crystal

structures at the FAD binding site, viewed from the solvent. The FAD and the FeS II are represented color-coded and correspond to the hAOX1

crystal structure. Note the drastic change in the hAOX1 loop T1230-Q1235 (hAOX1 numbering) when comparing to mAOX3 and bXO/bXDH proteins.

FAD


Thi-B

Thi-A

(R)-thioridazine

(S)-thioridazine

Supplementary Figure2: Bottom view of the surface representation of the hAOX1 homodimer highlighting the inhibitor thioridazine binding

pocket. Thioridazine is represented in space-filling-mode. At the bottom, the electron density of thioridazine is contoured at 1σ in the 2mFo–

DFc electron density map of the hAOX1 complex.


S100B_Chl(3lk0)

MALT1_Thi(4i1r)

PrP_Chl(4ma8)

hAOX1_Ph_Thi(4uhx, this work)

Pim1_Thi(4iaa)

P450_Thi (substrate)(4wnw)

Supplementary Figure3: Structures of complexes with phenothiazine-related drugs, reported in the PDB (top to bottom, left to right):

Complexes with inhibitors: S100B-p53 31 with chlorpromazine (3lk0); caspase-IG3 (human MALT1 complexed with Thi, (4i1r)30, Ser/Thr

kinase Pim1 with thioridazine (4iaa), prion protein PrP with chlorpromazine (4ma8)26, hAOX1_Ph_Thi (4uhx, this work). Complex with

substrate: Cytochrome P450 with thioridazine 32 (4wnw).


-0.05

0

0.05

0.1

0.15

-0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2

1/V

1/[phthalazine] (μM -1)

b)

0

0.01

0.02

0.03

0.04

0.05

-20 -10 0 10 20

1/V

max

ap

p

[ thioridazine] μM

-0.03

0.02

0.07

0.12

0.17

-0.7 -0.2 0.3 0.8

1/V

1/[xanthine] (μM -1)

c)

0

0.005

0.01

0.015

0.02

0.025

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

-450 -350 -250 -150 -50 50

Inte

rce

pt

(1/V

max

)

[ thioridazine] μM

-0.2

-0.1

0

0.1

0.2

0.3

0.4

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1

1/V

1/[phthalazine] (μM -1)

a)

0

0.01

0.02

-4 0 4 8 12 16 20

1/V

max

ap

p

[ thioridazine] μM

mAOX3 bXO

bXO:563:xxx*xx:*::* (…) :**:*::*::*:*K:::S::*

hAOX1:570IGPKQHPEDPIG (…) IQVVSRELRMPMSNVHLRGTS1076

mAOX3:570VDFQQPLQDPIG (…) IQVASRELKIPMSYIHLDEMS1072

bXO:563VPNGQSKEDTVG (…) VQVASKALKIPISKIYISETS1067

hAOX1

Supplementary Figure4: Kinetics of phthalazine-thioridazine inhibition of hAOX1 (a), mAOX3 (b) and xanthine-thioridazine inhibition of bXO (c).

Lineweaver-burk plots for substrate-electron acceptor reaction of hAOX1, mAOX3 and bXO in the presence of thioridazine. a) inhibition of 0.2–0.5

μM hAOX1 using 1, 1.5, 2.5, 3.75, 5, 10, 20, 40 µM phthalazine and 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15 μM thioridazine in the presence 1 mM

ferricyanide as electron acceptor. Inset: the Ki value was obtained from the secondary plot of the apparent 1/Vmax of the Linewaever-Burk plot. b)

inhibition of 0.2–0.5 µM mAOX3 using 1, 5, 10, 20, 40 µM phthalazine and 1, 5, 10, 15, 25 μM of thioridazine in the presence 1 mM ferricyanide as

electron acceptor. Inset: the Ki value was obtained from the secondary plot of the apparent 1/Vmax of the Linewaever-Burk plot. c) inhibition of 0.1

µM bXO using using 5, 10, 25, 10, 200 µM xanthine and 10, 25, 35, 50 μM thioridazine in the presence of molecular oxygen as electron acceptor.

Inset: the Ki and Kii values were obtained from the secondary plot of the slope apparent 1/Vmax of the Linewaever-Burk plot, respectively. Data are

mean values from three independent measurements (± S.D.) Alignment of the residues constituting the thioridazine binding pocket (d) and

structure of the surrounding region for hAOX1 (4uhx, in blue), mAOX3 (3zyv, in grey) and bXO (1fo4, in pink) (e).

d)

G580

I570

L1063e)


hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

hAOX1

mAOX1

mAOX3

mAOX2

mAOX4

GATE1

GATE2

600

l

620

l

640

l

660

l

700

l

720

l

740

l760

l

800

l820

l840

l

860

l

900

l

920

l

940

l960

l

1000

l

1020

l

1040

l1060

l

1100

l

1120

l

1140

l1160

l

1200

l

1220

l

1240

l1260

l

1300

l

1320

l

560

l

540

l

520

l

500

l

Supplementary Figure5: Comparison of

the amino acid sequence of human AOX1

and mouse AOX1, AOX3, AOX2 and AOX4

proteins. The hAOX1 Gate 1 and Gate 2

are highlighted in a red box and the

important active site residues are marked in

orange. The alignment was created with

using the clustalW2 server and represented

using the Jalview program.


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