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1 INHIBITION AND STRUCTURE OF TOXOPLASMA GONDII PURINE NUCLEOSIDE PHOSPHORYLASE Supplemental Material Teraya M. Donaldson a,¶ , María B. Cassera b,¶ , Meng-Chiao Ho b , Chenyang Zhan b , Emilio F. Merino b , Gary B. Evans c , Peter C. Tyler c , Steven C. Almo b , Vern L. Schramm b , and Kami Kim a. d, e # From the Departments of a Microbiology and Immunology, b Biochemistry, d Pathology and e Medicine, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461, USA. c Carbohydrate Chemistry Group, Callaghan Innovation, Lower Hutt, New Zealand Contributed equally to this study. Running title: Toxoplasma gondii purine nucleoside phosphorylase

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INHIBITION AND STRUCTURE OF TOXOPLASMA GONDII PURINE NUCLEOSIDE

PHOSPHORYLASE

Supplemental Material

Teraya M. Donaldsona,¶, María B. Casserab,¶, Meng-Chiao Hob, Chenyang Zhanb, Emilio F.

Merinob, Gary B. Evansc, Peter C. Tylerc, Steven C. Almob, Vern L. Schrammb, and Kami

Kima. d, e#

From the Departments of aMicrobiology and Immunology, bBiochemistry, dPathology and

eMedicine, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461,

USA.

cCarbohydrate Chemistry Group, Callaghan Innovation, Lower Hutt, New Zealand

¶Contributed equally to this study.

Running title: Toxoplasma gondii purine nucleoside phosphorylase

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TABLE S1. Buffer optimization for protein stability

Abbreviations: ADA - N-(2-Acetamido)iminodiacetic Acid, CAPS - 3-(Cyclohexylamino)-1-propanesulfonic acid, CAPSO - 3-(Cyclohexylamino)-2-hydroxy-1-propanesuhicic acid, CHES - N-Cyclohexyl-2-aminoethanesulfonic acid, EPPS - 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid, 4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid, HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, MES - 2-(N-morpholino)ethanesulfonic acid, MOPS - 3-(N-morpholino)propanesulfonic acid. PNP activity was not tested at 6 hours in samples that displayed little or no activity at the start of the stability experiment.

Buffer (salts) pH PNP activity (v0 at t=0, abs/min)

PNP activity (v0 at t=6 h, abs/min)

Average SD Average SD Sodium citrate 3.2 0.012 0.021 Sodium citrate 4.0 0.093 0.034 Sodium citrate 5.0 0.000 0.000 Sodium citrate 5.4 0.000 0.000 Sodium citrate 5.6 0.737 0.188 Sodium cacodylate 6.0 0.000 0.000 Sodium cacodylate 6.4 0.000 0.000 Sodium cacodylate 6.5 0.000 0.000 HEPES 7.0 0.000 0.000 HEPES 7.4 0.097 0.126 HEPES 7.5 0.695 0.134 Bicine 9.0 0.341 0.045 Na2HPO4/KH2PO4 5.0 3.703 0.058 2.97 0.35 Na2HPO4/KH2PO4 6.0 2.358 0.280 2.34 0.12 Na2HPO4/KH2PO4 7.0 0.644 0.057 0.47 0.08 Tris 7.5 0.947 0.061 0.00 0.05 Tris HCl 8.0 0.073 0.022 Tris HCl 8.5 0.000 0.000 Tris HCl 8.6 0.009 0.016 Bis Tris 5.5 0.023 0.040 Bis Tris 6.5 0.037 0.036 CHES 9.0 0.005 0.008 CHES 9.5 0.049 0.051 Imidazol 7.0 0.063 0.018 Imidazol 7.8 0.107 0.044 MES 6.5 0.569 0.039 Sodium acetate 4.0 0.000 0.000 Sodium acetate 4.6 0.000 0.000 Sodium acetate 4.5 0.546 0.026 Sodium acetate 7.0 2.238 0.119 1.99 0.13 CAPSO 9.0 0.068 0.059 ADA 6.5 1.225 0.061 0.00 0.03 MOPS 7.0 0.923 0.028 0.00 0.07 EPPS 8.0 0.004 0.008 CAPS 10.0 0.004 0.008

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TABLE S2. Comparison of inhibition constants for TgPNP, PfPNP, and HsPNP

aDissociation constant is defined as the tightest inhibition constant, either Ki or Ki

*. (*) Indicates Ki*.

bValues were previously reported by Lewandowicz et al. (24). Some values reported here differ from the

earlier report.

Dissociation constanta (nM)

Inhibitor TgPNP PfPNPb HsPNPb

Imm-H 0.37 ± 0.02 0.86 ± 0.08 0.06 ± 0.08 (*)

5´-d-Imm-H 11.0 ± 0.8 16 ± 1 12 ± 1 (*)

5´-F-Imm-H 20 ± 1 22 ± 1 (*) 4.6 ± 0.5 (*)

5´-COOH-Imm-H 120 ± 20 170 ± 9 > 50,000

2´-d-Imm-H 190 ± 24 19.0 ± 0.7 0.9 ± 0.1 (*)

DADMe-Imm-G 1,500 ± 300 0.90 ± 0.06 0.007 ± 0.001

DADMe-Imm-H 3,600 ± 720 0.50 ± 0.04 0.0085 ± 0.0002

5´-MT-Imm-H 5,600 ± 750 2.7 ± 0.4 300 ± 80

5´-CONH2-Imm-H 8,900 ± 300 220 ± 20 1,900 ± 50 (*)

5´-thio-Imm-H > 50,000 93 ± 8 (*) 86 ± 18 (*)

1´,9-Me-Imm-H > 50,000 > 600,000 250 ± 7

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Figure S1

Figure S1. Buffer optimization for protein stability. Immediately after purification, the recombinant protein was diluted 100-fold into 50 mM test buffer containing 50 mM NaCl and 1 mM dithiothreitol, and an aliquot from each condition was tested for purine nucleoside phosphorylase (PNP) activity as described in the Material and Methods section. The remaining protein was incubated for 6 hours at 4°C and retested for PNP activity.

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Figure S2

Figure S2: Stereo views of the catalytic site contacts in TgPNP electron density map with the transition sate analogue inhibitor Immucillin-H and PO4

3-. 2Fo-Fc map shows light blue side chains of the parental monomer surrounding the bound Immucillin-H (yellow); the green subunits on the bottom right indicate residues contributed from the adjacent subunit.

PO43-

Imm-H

Arg47b

His9b

Asp207

Trp213 Glycerol

PO43-

Imm-H

Arg47b

His9b

Asp207

Trp213 Glycerol