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 Variant

Amino acid change Structural context

Enzyme Activity

*1 WT

*2 A80P Helix Low

*3A A154T/Y240C b-Strand Low

*3B A154T b-Strand Low

*3C Y240C b-Strand Low

*3D Stopp/A154T/Y240C Low

*4 Splicing Low

*5 L49S Helix Low

*6 Y180F b-Strand Low

*7 H227Q Helix Low

*8 R215H b-Strand Intermediate

*9 K119T b-Strand Intermediate

*10 G144R Low

*11 C132Y b-Strand Low

*12 S125L Turn Low

*13 E28V Helix Low

*14 Deletion Low

*15 Deletion Low

*16 R163H Helix Intermediate

*17 Q42E Intermediate

*18 G71R Turn Intermediate

*19 K122T b-Strand High

*20 K238E b-Strand

*21 L69V Turn

*22 R163P Helix

*23 A167G Helix High/low??

*24 Q179H b-Strand

*25 C212R Turn

TPMT variants

Position of TPMT*2, *3C, *5 mutations

*3C (Tyr240→Cys) *2 (Ala80→Pro)

*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

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Resid

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icity

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ol-1

]

Wavelength [nm]

TPMT*1

TPMT*2

TPMT*3C

TPMT*5

Secondary structure analysis

Thermal unfolding analysis (222 nm)

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Temperature [°C] TPMT*1 TPMT*2 TPMT*3C TPMT*5

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

Thermodynamic parameters

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.

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resc

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GdnCl [M]

TPMT*1

TPMT*2

TPMT*3C

TPMT*5

ANS fluorescence measurements at 478 nm with a gradient of GdnCl

!

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Wavelength [nm]

0 µM SAM

100 µM SAM

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3 mM SAM

ANS fluorescence measurements of TPMT*1 at different concentrations of SAM

SAM/ANS binding site

ANS binding site

SAM/ANS binding site

ANS binding site

SAM/ANS binding site

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Wavelength [nm]

TPMT*1 no SAM

TPMT*2 no SAM

TPMT*3C no SAM

TPMT*5 no SAM

ANS fluorescent measurements

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%

Enzyme activity

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.

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TPMT*1

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TPMT*2

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TPMT*1

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915

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323

9

TPMT*5

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.

Thank you for listening