Structural Characteristics Determine the Cause of the Low Enzyme Activity of Two Thiopurine S-Methyltransferase Allelic Variants: A Biophysical Characterization of TPMT*2 and TPMT*5

Abstract

The enzyme thiopurine S-methyltransferase (TPMT) is involved in the metabolism of thiopurine drugs used to treat acute lymphoblastic leukemia and inflammatory bowel disease. Thus far, at least 29 variants of the TPMT gene have been described, many of which encode proteins that have low enzyme activity and in some cases become more prone to aggregation and degradation. Here, the two naturally occurring variants, TPMT*2 (Ala80 → Pro) and TPMT*5 (Leu49 → Ser), were cloned and expressed in Escherichia coli. Far-UV circular dichroism spectroscopy showed that TPMT*2 was substantially destabilized whereas TPMT*5 showed much greater stability comparable to that of wild-type TPMT (TPMTwt). The extrinsic fluorescent molecule anilinonaphthalene sulfonate (ANS) was used to probe the tertiary structure during thermal denaturation. In contrast to TPMTwt, neither of the variants bound ANS to a large extent. To explore the morphology of the TPMT aggregates, we performed luminescent conjugated oligothiophene staining and showed fibril formation for TPMT*2 and TPMT*5. The differences in the flexibility of TPMTwt, TPMT*2, and TPMT*5 were evaluated in a limited proteolysis experiment to pinpoint stable regions. Even though there is only one amino acid difference between the analyzed TPMT variants, a clear disparity in the cleavage patterns was observed. TPMT*2 displays a protected region in the C-terminus, which differs from TPMTwt, whereas the protected regions in TPMT*5 are located mainly in the N-terminus close to the active site. In conclusion, this in vitro study, conducted to probe structural changes during unfolding of TPMT*2 and TPMT*5, demonstrates that the various causes of the low enzyme activity in vivo could be explained on a molecular level.

MTMDAT-HADDOCK: High-throughput, protein complex structure modeling based on limited proteolysis and mass spectrometry

“…The resulting data can provide rather detailed information about protein interactions, stability and tertiary structure [11-17]. However, until recently, the extraction of this information was difficult due to the amount of data to be evaluated.…”

A comparative study of polyethylene glycol hydrogels derivatized with the RGD peptide and the cell-binding domain of fibronectin

“…Limited proteolysis and CD studies have been used extensively to characterize protein folding. [31][32][33][34] We added a chymotrypsin, which cleaves between fibronectin modules, 15 to native and PEG-conjugated GST-III 9-10 and characterized the products of proteolysis by SDS-PAGE. Figure 5(A) shows the products of a chymotrypsin proteolysis 5-10 min after protease addition.…”

Pharmacogenetic studies of thiopurine methyltransferase genotype-phenotype concordance and effect of methotrexate on thiopurine metabolism

“…The most common and studied effect of SNPs found in TPMT are single amino acid substitutions disturbing TPMT enzyme structure and stability [105,187,188]. Decreased quantities of TPMT protein have often been the explanation for low enzyme activity of defective TPMT alleles [15] and correlations between protein amount and enzyme activity for most TPMT*1-13 have been reported [184].…”