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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