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Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants

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Paolo Dametto
Paolo Dametto

This document describes biophysical studies of Thiopurine S-methyltransferase (TPMT) variants. TPMT is an enzyme that metabolizes thiopurine drugs used to treat conditions like leukemia. The document identifies several naturally occurring TPMT variants with reduced or intermediate enzyme activity. It then details experiments analyzing the structure, stability, and activity of wild type and variant TPMT proteins using techniques like circular dichroism, thermal unfolding, limited proteolysis, and enzyme activity assays. The results provide insights into how common TPMT variants alter the protein's structure and function, affecting thiopurine drug metabolism.

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Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants Paolo Dametto September 2009
TPMT ? TPMT (thiopurine S-methyltransferase) is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydril compounds, including drugs such as thiopurines.
Thiopurines 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and azathiopurine (AZA) are thiopurine drugs. ? lymphoblastic leukemia (ALL) ? inflammatory bowel disease (IBD) ? autoimmune disorders ? organ transplant recipient 6-MP 6-TG AZA
TPMT structure
TPMT variants TPMT Variant Amino acid change Structural context Enzyme Activity *1 WT *2 A80P Helix Low *3A A154T/Y240C b-Strand Low *14 Deletion Low *3B A154T b-Strand Low *15 Deletion Low *3C Y240C b-Strand Low *16 R163H *3D Stopp/A154T/Y240C Low *4 Splicing Low *17 Q42E *5 L49S Helix Low *18 G71R Turn Intermediate *6 Y180F b-Strand Low *19 K122T b-Strand High *7 H227Q Helix Low *20 K238E b-Strand *8 R215H b-Strand Intermediate *21 L69V Turn *22 R163P Helix *23 A167G Helix b-Strand Helix Intermediate Intermediate *9 K119T Intermediate *10 G144R *11 C132Y b-Strand Low *12 S125L Turn Low *24 Q179H b-Strand *13 E28V Helix Low *25 C212R Turn Low High/low??
Position of TPMT*2, *3C, *5 mutations *2 (Ala80★Pro) *3C (Tyr240★Cys) *5 (Leu49★Ser)
Purification¨s step´´ 1. TPMT large scale expression in BL21/DE3 cells 2. Sonication 3. His-Tag purification using Ni-NTA column 4. Removing His-Tag sequence with Biotinylated Thrombin 5. His-Tag purification 6. Gel Filtration chromatography
CD: circular dichroism The FAR-UV (190 ‖ 260 nm) CD spectrum can reveal important characteristics of proteins. ? estimation of secondary structure ? molecule changes in the secondary structure as a function of temperature or of the concentration of denaturing agents ? thermodynamic information such as ?G and Tm
Secondary structure analysis 35000 Residue ellipticity [degrees*cm2*dmol-1] 30000 TPMT*1 25000 TPMT*2 20000 TPMT*3C 15000 TPMT*5 10000 5000 0 -5000 190 200 210 220 230 -10000 -15000 -20000 -25000 -30000 Wavelength [nm] 240 250 260 270
Thermal unfolding analysis (222 nm) 10 15 20 25 30 35 40 45 50 55 60 65 Residue ellipticity [degrees*cm2*dmol-1] 0 -1000 -2000 -3000 -4000 -5000 -6000 -7000 -8000 -9000 -10000 -11000 -12000 -13000 Temperature [＜C] TPMT*1 TPMT*2 TPMT*3C TPMT*5 70 75 80 85
Thermodynamic parameters From Thermal Denaturation Analysis FAR UV_222nm Protein Tm [＜C] ?G(25＜C) [Kcal/mol] ?(?G) [Kcal/mol] ?Sm [J/mol*K] ?Hm [Kcal/mol] TPMT*1 58.9 14.5 // -1788.4 141.9 TPMT*2 32.8 1.27 11.19 -549.9 40.2 TPMT*3C 42.9 3.77 6.84 -879.8 66.4 TPMT*5 49.3 7.66 4.14 -1318.7 101.6
ANS ?The aromatic chromophore 1-anilino-8-naphthalene sulfonate (ANS) is feebly fluorescent in water but the intensity is dramatically increased in nonpolar solvent or when it binds to nonpolar sites of proteins. ?ANS was used to check if the TPMT variants showed some hydrophobic pattern, according to their enhanced thermodynamic instability. In this case, the ANS signal would be stronger than that of TPMT*1.
ANS fluorescence measurements at 478 nm with a gradient of GdnCl 12500 11500 ! 10500 Fluorescence Intensity 9500 8500 TPMT*1 TPMT*2 TPMT*3C 7500 TPMT*5 6500 5500 4500 3500 2500 1500 500 -500 0 0.25 0.5 0.75 1 1.25 1.5 GdnCl [M] 1.75 2 2.25 2.5 2.75
Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants
Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants
Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants
Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants
ANS fluorescence measurements of TPMT*1 at different concentrations of SAM 10500000 0 ?M SAM 100 ?M SAM 8500000 200 ?M SAM 7500000 Fluorescence intesity 9500000 1 mM SAM 6500000 3 mM SAM 5500000 4500000 3500000 2500000 1500000 500000 -500000 400 420 440 460 480 500 Wavelength [nm] 520 540 560 580 600
SAM/ANS binding site
ANS binding site SAM/ANS binding site
ANS binding site SAM/ANS binding site
ANS fluorescent measurements 11500000 10500000 TPMT*1 no SAM 8500000 Fluorescence intensity 9500000 TPMT*2 no SAM TPMT*3C no SAM 7500000 TPMT*5 no SAM 6500000 5500000 4500000 3500000 2500000 1500000 500000 -500000 400 420 440 460 480 500 520 Wavelength [nm] 540 560 580 600
Enzyme activity Protein Enzyme activity at 37 ＜C Enzyme activity at 18 ＜C TPMT *1 (WT) 100% 100% TPMT *2 (A80P) 35% 48% TPMT *5 (L49S) 0% 14%
Limited proteolysis Limited proteolysis can be used to probe conformational features of protein. ? Limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) was applied to probe protease-accessible sites of TPMT. ? Fragments were analyzed using the software MTMDAT.
234 227 220 213 206 199 192 185 178 171 164 157 150 143 136 129 122 115 108 101 94 87 80 73 66 59 52 45 38 31 24 17 0.15 0.1 0.15 0.1 235 228 221 214 207 200 193 186 179 171 164 157 150 143 136 129 122 115 108 101 94 87 80 73 66 59 52 45 38 31 24 17 0.3 0.25 0.2 TPMT*1 0.05 0 0 0.3 0.25 0.2 TPMT*2 0.05 0 0
0.1 239 191 185 221 215 209 239 0.1 233 167 197 227 155 161 83 0.15 233 0.15 227 221 215 209 203 137 131 125 143 173 107 113 59 65 77 95 47 41 17 23 29 35 53 71 89 101 119 149 179 203 0.3 197 191 185 179 173 167 161 155 149 143 137 131 125 119 113 107 101 95 89 83 77 71 65 59 53 47 41 35 29 23 17 0.3 0.25 0.2 TPMT*1 0.05 0 0 0.25 0.2 TPMT*5 0.05 0 0
TPMT*2 TPMT*5
Considerations ? The TPMT protein can broaden our understanding of the mechanisms by which common polymorphisms can lead to functional effects. ? TPMT protein represents one of the most striking example of the science named Pharmacogenomics.
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Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants