In this work, we used Small-angle X-ray and neutron scattering (SAS) to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa (P.aeruginosa) transcriptional regulator MexR, a member of the MarR family, when bound to one of its native DNA binding sites. Several MarR-like proteins, including MexR, repress the expression of efflux pump proteins by binding to DNA on regulatory sites overlapping with promoter regions. When expressed, efflux-proteins self-assemble to form multiprotein complexes and actively expel highly toxic compounds out of the host organism. The mutational pressure on efflux-regulating MarR family proteins is high since deficient DNA binding leads to constitutive expression of efflux pumps and thereby supports acquired multidrug resistance. Understanding the functional outcome of such mutations and their effects on DNA binding has been hampered by the scarcity of structural and dynamic characterisation of both free and DNA-bound MarR proteins. Here, we show how combined neutron and X-ray small-angle scattering (SAS) of both states in solution support a conformational selection model that enhances MexR asymmetry in binding to one of its promoter-overlapping DNA binding sites.

The discovery and implementation of thiopurine methyltransferase (TPMT) pharmacogenetics has been a success story and has reduced the suffering from serious adverse reactions during thiopurine treatment of childhood leukaemia and inflammatory bowel disease. This MiniReview summarizes four studies included in Dr Zimdahl Kahlin's doctoral thesis as well as the current knowledge on this field of research. The genotype-phenotype concordance of TPMT in a cohort of 12 663 individuals with clinically analysed TPMT status is described. Notwithstanding the high concordance, the benefits of combined genotyping and phenotyping for TPMT status determination are discussed. The results from the large cohort also demonstrate that the factors of gender and age affect TPMT enzyme activity. In addition, characterization of four previously undescribed TPMT alleles (TPMT*41, TPMT*42, TPMT*43 and TPMT*44) shows that a defective TPMT enzyme could be caused by several different mechanisms. Moreover, the folate analogue methotrexate (MTX), used in combination with thiopurines during maintenance therapy of childhood leukaemia, affects the metabolism of thiopurines and interacts with TPMT, not only by binding and inhibiting the enzyme activity but also by regulation of its gene expression.

Thiopurine S-methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism and inactivation of thiopurine substances administered as immunosuppressants in the treatment of malignancies and autoimmune diseases. In this study, the naturally occurring variants, TPMT*6 (Y180F) and TPMT*8 (R215H), have been biophysically characterized. Despite being classified as low and intermediate in vivo enzyme activity variants, respectively, our results demonstrate a discrepancy because both TPMT*6 and TPMT*8 were found to exhibit normal functionality in vitro. While TPMT*8 exhibited biophysical properties almost indistinguishable from those of TPMTwt, the TPMT*6 variant was found to be destabilized. Furthermore, the contributions of the cofactor S-adenosylmethionine (SAM) to the thermodynamic stability of TPMT were investigated, but only a modest stabilizing effect was observed. Also presented herein is a new method for studies of the biophysical characteristics of TPMT and its variants using the extrinsic fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS). ANS was found to bind strongly to all investigated TPMT variants with a K_d of approximately 0.2 μM and a 1:1 binding ratio as determined by isothermal titration calorimetry (ITC). Circular dichroism and fluorescence measurements showed that ANS binds exclusively to the native state of TPMT, and binding to the active site was confirmed by molecular modeling and simulated docking as well as ITC measurements. The strong binding of the probe to native TPMT and the conformity of the obtained results demonstrate the advantages of using ANS binding characteristics in studies of this protein and its variants.

Thiopurines are chemotherapeutic drugs used for treatment of inflammatory bowel diseases and childhood leukemia. Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurines. Individuals lacking TPMT are at increased risk for severe side effects when treated with conventional doses of thiopurines. A research group at the division of drug research at Linköping University is studying thiopurine pharmacogenetics. Since the year 2000, the lab has determined the TPMT status in over 12000 individuals, as an aid to decide thiopurine doses before starting treatment. New knowledge of how genetic factors influence thiopurine treatment effect are anticipated to improve the possibilities for individualization of thiopurine therapy.

We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.

Purpose

Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients.

Methods

Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line.

Results

Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (−9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %).

Conclusions

Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.

BACKGROUND: The activity of the human enzyme thiopurine methyltransferase (TPMT) varies greatly between individuals because of genetic polymorphism. TPMT is involved in the detoxification and activation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathioprine. These drugs are used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disease. A total of 29 sequence variants have been identified so far in the TPMT gene. However, most of these variants are rare and not fully characterized. METHODS AND RESULTS: In this study, we describe the identification and characterization of a novel TPMT sequence variant, originally found in a Swedish man of Italian origin. Sequencing of the variable number tandem repeats region of the TPMT promoter and exons III-X revealed a T-to-C transition at nucleotide 611, causing an amino acid substitution from isoleucine to threonine at amino acid 204, positioned in an α-helix, approximately 16 Å from the active site. This new variant was found in the patient and in his son. Both had intermediate enzyme activity (8.1 U/ml packed red blood cells and 8.8 U/ml packed red blood cells, respectively) and neither carried other variants in the coding region of the gene. To be able to study this variant in more detail, the TPMT*28 variant was expressed in Escherichia coli, and an in-vitro characterization of the variant revealed that the protein was destabilized and showed a stronger tendency towards degradation at 37°C than the wild-type protein. The individuals carrying the TPMT*28 variant had less TPMT protein and lower TPMT activity in both red and white blood cells compared with a wild-type control. CONCLUSIONS: We present a detailed in-vivo and in-vitro characterization of a novel TPMT sequence variant (TPMT*28) causing decreased TPMT activity. Individuals carrying TPMT*28 might have an increased risk for developing severe side effects if treated with conventional doses of thiopurines.

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.

In treatment of acute lymphoblastic leukemia and inflammatorybowel disease (IBD) thiopurines such as azathioprine and 6-mercaptopurineare used. All of these drugs are prodrugs and are, inthe cell, converted to 6-thioguanines (6-TGNs) and incorporatedinto DNA or inhibiting purine synthesis. A key enzyme for thisregulation is the cytosolic enzyme thiopurine S-methyltransferase(TPMT). This enzyme degrades azathioprine and 6-mercaptopurineto methylmercapto-purine and thereby reduces the bioavailabilityof the 6-TGNs incorporated into DNA. TPMT is apolymorphic enzyme with at least 29 different allelic variantsknown today and is one of the more classical examples of pharmacogeneticswhere the TPMT enzyme activity of the allelic variantsis directly correlated to the clinical dosages of the thiopurines, with a 10–15 fold dosage reduction for an allelic variantwith low TPMT enzyme activity. Even though TPMT is awell studied protein. Many studies have been performed in yeast‘‘suspensions’’ and not on pure protein solutions. It has beenspeculated and in a few cases shown that the reason for the lowactivity for most of the allelic variants is mainly due to the lowstability and/or tendency to aggregate. The mutations in thisstudy TPMT *2 (A80P) and TPMT * 5 (L49S) are both situatedat a distance far from the active site, however the enzyme activitiesare severely affected at 37°C. Preliminary results, using a repertoireof techniques such as CD, fluorescence and limitedproteolysis experiments suggest two different mechanisms for thelow enzyme activity at a temperature corresponding to in vivo conditions.

Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurine drugs. Owing to polymorphisms in the TPMT gene (TPMT*2-*22), the enzyme activity varies interindividually. Patients with reduced TPMT activity may develop adverse reactions when treated with standard doses of thiopurines. This work focuses on a TPMT genotype/phenotype discrepancy found in a patient during routine testing. The patient displayed very low TPMT enzyme activity and she was genotyped by pyrosequencing as being heterozygous for the 460G>A and 719A>G polymorphisms (TPMT*3A). Complete sequencing in combination with haplotyping of the TPMT gene revealed a novel sequence variant, 500C>G, on one allele and TPMT*3A on the other allele, giving rise to the novel genotype TPMT*3A/*23. When investigating the patient's relatives, they too had the TPMT*3A/*23 genotype in combination with low enzyme activity. We conclude that this novel variant allele affects enzyme activity, as the individuals carrying it had almost undetectable TPMT activity.