Small-angle x-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding model.

In this work, we used small-angle x-ray and neutron scattering to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa transcriptional regulator MexR, a member of the multiple antibiotics resistance regulator (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 characterization of both free and DNA-bound MarR proteins. Here, we show how combined neutron and x-ray small-angle scattering 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.

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

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.

Comprehensive study of thiopurine methyltransferase genotype, phenotype, and genotype-phenotype discrepancies in Sweden.

Thiopurines are widely used in the treatment of leukemia and inflammatory bowel diseases. Thiopurine metabolism varies among individuals because of differences in the polymorphic enzyme thiopurine methyltransferase (TPMT, EC 2.1.1.67), and to avoid severe adverse reactions caused by incorrect dosing it is recommended that the patient's TPMT status be determined before the start of thiopurine treatment. This study describes the concordance between genotyping for common TPMT alleles and phenotyping in a Swedish cohort of 12,663 patients sampled before or during thiopurine treatment. The concordance between TPMT genotype and enzyme activity was 94.5%. Compared to the genotype, the first measurement of TPMT enzyme activity was lower than expected for 4.6% of the patients. Sequencing of all coding regions of the TPMT gene in genotype/phenotype discrepant individuals led to the identification of rare and novel TPMT alleles. Fifteen individuals (0.1%) with rare or novel genotypes were identified, and three TPMT alleles (TPMT*42, *43, and *44) are characterized here for the first time. These 15 patients would not have been detected as carrying a deviating TPMT genotype if only genotyping of the most common TPMT variants had been performed. This study highlights the benefit of combining TPMT genotype and phenotype determination in routine testing. More accurate dose recommendations can be made, which might decrease the number of adverse reactions and treatment failures during thiopurine treatment.

Methylation of selenocysteine catalysed by thiopurine S-methyltransferase.

BACKGROUND: Methylation driven by thiopurine S-methylatransferase (TPMT) is crucial for deactivation of cytostatic and immunosuppressant thiopurines. Despite its remarkable integration into clinical practice, the endogenous function of TPMT is unknown. METHODS: To address the role of TPMT in methylation of selenium compounds, we established the research on saturation transfer difference (STD) and 77Se NMR spectroscopy, fluorescence measurements, as well as computational molecular docking simulations. RESULTS: Using STD NMR spectroscopy and fluorescence measurements of tryptophan residues in TPMT, we determined the binding of selenocysteine (Sec) to human recombinant TPMT. By comparing binding characteristics of Sec in the absence and in the presence of methyl donor, we confirmed S-adenosylmethionine (SAM)-induced conformational changes in TPMT. Molecular docking analysis positioned Sec into the active site of TPMT with orientation relevant for methylation reaction. Se-methylselenocysteine (MeSec), produced in the enzymatic reaction, was detected by 77Se NMR spectroscopy. A direct interaction between Sec and SAM in the active site of rTPMT and the formation of both products, MeSec and S-adenosylhomocysteine, was demonstrated using NMR spectroscopy. CONCLUSIONS: The present study provides evidence on in vitro methylation of Sec by rTPMT in a SAM-dependant manner. GENERAL SIGNIFICANCE: Our results suggest novel role of TPMT and demonstrate new insights into enzymatic modifications of the 21st amino acid.

One amino acid makes a difference-Characterization of a new TPMT allele and the influence of SAM on TPMT stability.

Thiopurine induced toxicity is associated with defects in the thiopurine methyltransferase (TPMT) gene. TPMT is a polymorphic enzyme, with most of the single nucleotide polymorphisms (SNPs) causing an amino acid change, altering the enzymatic activity of the TPMT protein. In this study, we characterize a novel patient allele c.719A > C, named TPMT*41, together with the more common variant *3C c.719A > G, resulting in an amino acid shift at tyrosine 240 to serine, p.Y240S and cysteine, p.Y240C respectively. We show that the patient heterozygote for c.719A > C has intermediate enzymatic activity in red blood cells. Furthermore, in vitro studies, using recombinant protein, show that TPMT p.Y240S is less stable than both TPMTwt and TPMT p.Y240C. The addition of SAM increases the stability and, in agreement with Isothermal Titration Calorimetry (ITC) data, higher molar excess of SAM is needed in order to stabilize TPMT p.Y240C and TPMT p.Y240S compared to TPMTwt. Molecular dynamics simulations show that the loss of interactions is most severe for Y240S, which agrees with the thermal stability of the mutations. In conclusion, our study shows that SAM increases the stability of TPMT and that changing only one amino acid can have a dramatic effect on TPMT stability and activity.

In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT*6 and TPMT*8.

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 Kd 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.

Robust and convenient analysis of protein thermal and chemical stability.

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.

[The usefulness of pharmacogenetics for a more individualized treatment. The example thiopurines in inflammatory bowel disease and childhood leukemia]. / Nyttan av farmakogenetik för en mer individualiserad behandling - Exemplet tiopuriner vid inflammatorisk tarmsjukdom och barnleukemi.

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.

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia.

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.

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.

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.

Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene.

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.

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance.

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.

Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.

UNLABELLED: Marble brain disease (MBD) also known as Guibaud-Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded beta-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619-633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. IN CONCLUSION: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.

Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains.

The effect of modulating the shape and the size of the hydrophobic pocket on the esterase activity and specificity of human carbonic anhydrase II (HCAII) for esters with different acyl chain lengths was investigated. Following an initial screen of 7 HCAII variants with alanine substitutions in positions 121, 143 and 198, detailed kinetic measurements were performed on HCAII and the variants V121A, V143A and V121A/V143A. For some variants, an increased size of the hydrophobic pocket resulted in increased activities and specificities for longer substrates. For V121A/V143A, the rate of hydrolysis for paranitrophenyl valerate was increased by a factor of approximately 3000. The specificities also changed dramatically, for example V121A/V143A is 6.3 times more efficient with paranitrophenyl valerate than paranitrophenyl acetate, while HCAII is >500 times more efficient with paranitrophenyl acetate than paranitrophenyl valerate. An automated docking procedure was performed on these variants with transition state analogues (TSAs) for the hydrolysis reaction. It was possible to correlate the catalytic rate constants to the docking results, i.e. for each variant, efficient hydrolysis was generally correlated to successful TSA-docking. The observations in this paper show that the redesign increased the catalytic rates for substrates with long acyl chains by removal of steric hinders and addition of new favourable binding interactions.

Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations.

The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).

Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase.

The native state of the enzyme human carbonic anhydrase (HCA II) has been stabilized by the introduction of a disulfide bond, the oxidized A23C/L203C mutant. This stabilized protein variant undergoes an apparent two-state unfolding process with suppression of the otherwise stable equilibrium, molten-globule intermediate, which is normally very prone to aggregation. Stopped-flow measurements also showed that lower amounts of the transiently occurring molten globule were formed during refolding. This led to a markedly lowered tendency for aggregation during equilibrium denaturing conditions and, more importantly, to significantly higher reactivation yields upon refolding of the fully denatured protein. Thus, a general strategy to circumvent aggregation during the refolding of proteins could be to stabilize the native state of a protein at the expense of partially folded intermediates, thereby shifting the unfolding behavior from a three-state process to a two-state one.

Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond.

To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten-globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.

A test of proposed rules for helix capping: implications for protein design.

alpha-helices within proteins are often terminated (capped) by distinctive configurations of the polypeptide chain. Two common arrangements are the Schellman motif and the alternative alpha(L) motif. Rose and coworkers developed stereochemical rules to identify the locations of such motifs in proteins of unknown structure based only on their amino acid sequences. To check the effectiveness of these rules, they made specific predictions regarding the structural and thermodynamic consequences of certain mutations in T4 lysozyme. We have constructed these mutants and show here that they have neither the structure nor the stability that was predicted. The results show the complexity of the protein-folding problem. Comparison of known protein structures may show that a characteristic sequence of amino acids (a sequence motif) corresponds to a conserved structural motif. In any particular protein, however, changes in other parts of the sequence may result in a different conformation. The structure is determined by sequence as a whole, not by parts considered in isolation.