SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations.

BACKGROUND: Half of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1. METHODS AND RESULTS: By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced. CONCLUSIONS: SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53. GENERAL SIGNIFICANCE: This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.

Spatial requirement for PAMO for transformation of non-native linear substrates.

Phenylacetone monooxygenase is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenases family, and therefore it is an ideal candidate for the synthesis of industrially relevant ester or lactone compounds. However, its limited substrate scope has largely limited its industrial applications. Linear substrates are interesting from an industrial point of view, it is thus necessary to identify the essential spatial requirement for achieving high conversions for non-native linear substrates. Here using molecular dynamics simulations, we compared the conversion of a non-native linear substrate 2-octanone and the native substrate phenylacetone, catalyzed by the WT enzyme and a quadruple variant P253F/G254A/R258M/L443F that exhibits significantly improved activity towards 2-octanone. We uncovered that a remarkable movement of L289 is crucial for a reshaping of the active site of the quadruple variant so as to prevent the aliphatic substrate from moving away from the C4a-peroxyflavin, thus enabling it to keep a catalytically relevant pose during the oxygenation process. By performing steady-state kinetic analysis of two single-mutation variants at position 258, we further validated that the L289 reposition is attributed to the combined effect of quadruple mutations. In order to further explore the substrate scope of PAMO we also studied the binding of cyclopentanone and 2-phenylcyclohexanone, which are the typical substrates of CPMO in group I and CHMO in group III, respectively. Our study provides fundamental atomic-level insights in rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.

Why the Flavin Adenine Dinucleotide (FAD) Cofactor Needs To Be Covalently Linked to Complex†II of the Electron-Transport Chain for the Conversion of FADH2 into FAD.

A covalently bound flavin cofactor is predominant in the succinate-ubiquinone oxidoreductase (SQR; Complex II), an essential component of aerobic electron transport, and in the menaquinol-fumarate oxidoreductase (QFR), the anaerobic counterpart, although it is only present in approximately 10 % of the known flavoenzymes. This work investigates the role of this 8α-N3-histidyl linkage between the flavin adenine dinucleotide (FAD) cofactor and the respiratory Complex II. After parameterization with DFT calculations, classical molecular-dynamics simulations and quantum-mechanics calculations for Complex II:FAD and Complex II:FADH2 , with and without the covalent bond, were performed. It was observed that the covalent bond is essential for the active-center arrangement of the FADH2 /FAD cofactor. Removal of this bond causes a displacement of the isoalloxazine group, which influences interactions with the protein, flavin solvation, and possible proton-transfer pathways. Specifically, for the noncovalently bound FADH2 cofactor, the N1 atom moves away from the His-A365 and His-A254 residues and the N5 atom moves away from the glutamine-62A residue. Both of the histidine and glutamine residues interact with a chain of water molecules that cross the enzyme, which is most likely involved in proton transfer. Breaking this chain of water molecules could thereby compromise proton transfer across the two active sites of Complex II.

Catalytic mechanism of phenylacetone monooxygenases for non-native linear substrates.

Phenylacetone monooxygenase (PAMO) is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenase family, and therefore it is an ideal candidate for the synthesis of industrially relevant compounds. However, its limited substrate scope has largely limited its industrial applications. In the present work, we provide, for the first time, the catalytic mechanism of PAMO for the native substrate phenylacetone as well as for a linear non-native substrate 2-octanone, using molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations. We provide a theoretical basis for the preference of the enzyme for the native aromatic substrate over non-native linear substrates. Our study provides fundamental atomic-level insights that can be employed in the rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.

Insights from engineering the Affibody-Fc interaction with a computational-experimental method.

The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes. We used a variation of FoldX to predict changes in protein-protein-binding affinity, and produce a list of 41 single-amino acid substitutions on the SpA molecule, of which four are near wild type (wt) and five are at most a factor of four from wt affinity. The nine substitutions include one which removes lysine, and several others which change charge. Subtle modulations in affinity may be useful for modifying column elution conditions. The method is applicable to other protein-protein systems, providing molecular insights with lower workload than existing experimental techniques.

Modeling and fitting protein-protein complexes to predict change of binding energy.

It is possible to accurately and economically predict change in protein-protein interaction energy upon mutation (ΔΔG), when a high-resolution structure of the complex is available. This is of growing usefulness for design of high-affinity or otherwise modified binding proteins for therapeutic, diagnostic, industrial, and basic science applications. Recently the field has begun to pursue ΔΔG prediction for homology modeled complexes, but so far this has worked mostly for cases of high sequence identity. If the interacting proteins have been crystallized in free (uncomplexed) form, in a majority of cases it is possible to find a structurally similar complex which can be used as the basis for template-based modeling. We describe how to use MMB to create such models, and then use them to predict ΔΔG, using a dataset consisting of free target structures, co-crystallized template complexes with sequence identify with respect to the targets as low as 44%, and experimental ΔΔG measurements. We obtain similar results by fitting to a low-resolution Cryo-EM density map. Results suggest that other structural constraints may lead to a similar outcome, making the method even more broadly applicable.

A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B.

Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. α-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to α-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of α-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by α-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal α-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to α-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-α-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with α-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in α-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.

Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin.

Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.

Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates.

BACKGROUND: Organic isothiocyanates (ITCs) are produced by plants, in which they are released from glucosinolates by myrosinase. ITCs are generally toxic and serve as a chemical defense against herbivorous insects and against infections by microorganisms. In mammalian tissues subtoxic concentrations of ITCs can provide protective effects against cancer and other diseases partially by induction of glutathione transferases (GSTs) and other detoxication enzymes. Thus, human consumption of edible plants rich in ITCs is presumed to provide health benefits. ITCs react with intracellular glutathione to form dithiocarbamates, catalyzed by GSTs. Formation of glutathione conjugates is central to the biotransformation of ITCs and leads to a route for their excretion. Clearly, the emergence of ITC conjugating activity in GSTs is essential from the biological and evolutionary perspective. METHODS: In the present investigation an active-site-focused mutant library of GST A2-2 has been screened for enzyme variants with enhanced ITC activity. RESULTS: Significantly superior activities were found in 34 of the approximately 2000 mutants analyzed, and the majority of the superior GSTs featured His and Gly residues in one of the three active-site positions subjected to mutagenesis. CONCLUSIONS: We explored the propensity of GSTs to obtain altered substrate selectivity and moreover, identified a specific pattern of mutagenesis in GST for enhanced PEITC detoxification, which may play an important role in the evolution of adaptive responses in organisms subjected to ITCs. GENERAL SIGNIFICANCE: The facile acquisition of enhanced ITC activity demonstrates that this important detoxication function can be promoted by numerous evolutionary trajectories in sequence space.

Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

A multiscale approach to predicting affinity changes in protein-protein interfaces.

Substitution mutations in protein-protein interfaces can have a substantial effect on binding, which has consequences in basic and applied biomedical research. Experimental expression, purification, and affinity determination of protein complexes is an expensive and time-consuming means of evaluating the effect of mutations, making a fast and accurate in silico method highly desirable. When the structure of the wild-type complex is known, it is possible to economically evaluate the effect of point mutations with knowledge based potentials, which do not model backbone flexibility, but these have been validated only for single mutants. Substitution mutations tend to induce local conformational rearrangements only. Accordingly, ZEMu (Zone Equilibration of Mutants) flexibilizes only a small region around the site of mutation, then computes its dynamics under a physics-based force field. We validate with 1254 experimental mutants (with 1-15 simultaneous substitutions) in a wide variety of different protein environments (65 protein complexes), and obtain a significant improvement in the accuracy of predicted ΔΔG.

New in silico insights into the inhibition of RNAP II by α-amanitin and the protective effect mediated by effective antidotes.

Poisonous α-amanitin-containing mushrooms are responsible for the major cases of fatalities after mushroom ingestion. α-Amanitin is known to inhibit the RNA polymerase II (RNAP II), although the underlying mechanisms are not fully understood. Benzylpenicillin, ceftazidime and silybin have been the most frequently used drugs in the management of α-amanitin poisoning, mostly based on empirical rationale. The present study provides an in silico insight into the inhibition of RNAP II by α-amanitin and also on the interaction of the antidotes on the active site of this enzyme. Docking and molecular dynamics (MD) simulations combined with molecular mechanics-generalized Born surface area method (MM-GBSA) were carried out to investigate the binding of α-amanitin and three antidotes benzylpenicillin, ceftazidime and silybin to RNAP II. Our results reveal that α-amanitin should affects RNAP II transcription by compromising trigger loop (TL) function. The observed direct interactions between α-amanitin and TL residues Leu1081, Asn1082, Thr1083, His1085 and Gly1088 alters the elongation process and thus contribute to the inhibition of RNAP II. We also present evidences that α-amanitin can interact directly with the bridge helix residues Gly819, Gly820 and Glu822, and indirectly with His816 and Phe815. This destabilizes the bridge helix, possibly causing RNAP II activity loss. We demonstrate that benzylpenicillin, ceftazidime and silybin are able to bind to the same site as α-amanitin, although not replicating the unique α-amanitin binding mode. They establish considerably less intermolecular interactions and the ones existing are essential confine to the bridge helix and adjacent residues. Therefore, the therapeutic effect of these antidotes does not seem to be directly related with binding to RNAP II. RNAP II α-amanitin binding site can be divided into specific zones with different properties providing a reliable platform for the structure-based drug design of novel antidotes for α-amatoxin poisoning. An ideal drug candidate should be a competitive RNAP II binder that interacts with Arg726, Ile756, Ala759, Gln760 and Gln767, but not with TL and bridge helix residues.

Isomerization of Δ5-androstene-3,17-dione into Δ4-androstene-3,17-dione catalyzed by human glutathione transferase A3-3: a computational study identifies a dual role for glutathione.

Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Δ5-androstene-3,17-dione (Δ(5)-AD) and Δ(5)-pregnene-3,20-dione (Δ(5)-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Δ(5)-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Δ(5)-AD C4 atom to the Δ(5)-AD C6 atom, with a rate limiting activation energy of 15.8 kcal · mol(-1). GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the O3 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leu111, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Δ(4)-AD crystal structure and is consistent with all available experimental data.

Mechanism of glutathione transferase P1-1-catalyzed activation of the prodrug canfosfamide (TLK286, TELCYTA).

Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO(-) groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.

Multidimensional epistasis and fitness landscapes in enzyme evolution.

The conventional analysis of enzyme evolution is to regard one single salient feature as a measure of fitness, expressed in a milieu exposing the possible selective advantage at a given time and location. Given that a single protein may serve more than one function, fitness should be assessed in several dimensions. In the present study we have explored individual mutational steps leading to a triple-point-mutated human GST (glutathione transferase) A2-2 displaying enhanced activity with azathioprine. A total of eight alternative substrates were used to monitor the diverse evolutionary trajectories. The epistatic effects of the mutations on catalytic activity were variable in sign and magnitude and depended on the substrate used, showing that epistasis is a multidimensional quality. Evidently, the multidimensional fitness landscape can lead to alternative trajectories resulting in enzymes optimized for features other than the selectable markers relevant at the origin of the evolutionary process. In this manner the evolutionary response is robust and can adapt to changing environmental conditions.

Glutathione transferase classes alpha, pi, and mu: GSH activation mechanism.

Since the early 1960s, glutathione transferases (GSTs) have been described as detoxification enzymes. In fact, GSTs are the most important enzymes involved in the metabolism of electrophilic xenobiotic/endobiotic compounds. These enzymes are able to catalyze the nucleophilic addition of glutathione (GSH) sulfur thiolate to a wide range of electrophilic substrates, building up a less toxic and more soluble compound. Cytosolic classes alpha, pi, and mu are the most extensively studied GSTs. However, many of the catalytic events are still poorly understood. In the present work, we have resorted to density functional theory (DFT) and to potential of mean force (PMF) calculations to determine the GSH activation mechanism of GSTP1-1 and GSTM1-1 isoenzymes. For the GSTP1-1 enzyme, we have demonstrated that a water molecule, after an initial conformational rearrangement of GSH, can assist a proton transfer between the GSH cysteine thiol (GSH-SH) and the GSH glutamate alpha carboxylate (GSH-COO(-)) groups. The energy barrier associated with the proton transfer is 11.36 kcal·mol(-1). The GSTM1-1 enzyme shows a completely different behavior from the previous isoenzyme. In this case, two water molecules, positioned between the GSH-SH and the ξ N atom of His107, working like a bridge, are able to promote the proton transfer between these two active groups with an energy barrier of 7.98 kcal·mol(-1). All our results are consistent with all the enzymes kinetics and mutagenesis experimental studies.

Glutathione transferase A1-1: catalytic importance of arginine 15.

Glutathione transferases (GSTs) are fundamental enzymes of the cell detoxification system. They catalyze the nucleophilic attack of glutathione (GSH) on electrophilic substrates to produce less toxic compounds. The resulting substrate can then be recognized by ATP-dependent transmembrane pumps and consequently expelled from the cell. Despite all the existing studies on GSTs, many aspects of the catalytic events are still poorly understood. Recently, using as a model the GSTA1-1 enzyme, we proposed a GSH activation mechanism. Resorting to the density functional theory (DFT), we demonstrated that a water molecule could assist a proton transfer between the GSH thiol and alpha-carboxylic groups, after an initial conformational rearrangement of GSH, as evidenced by potential of mean force calculations. In this work to elucidate the catalytic role of Arg15, a strictly conserved active site residue in class alpha GSTs, we analyzed the activation energy barrier and structural details associated with the GSTA1-1 mutants R15A, R15Repsilon,eta-c (an Arg residue with the epsilon,eta-nitrogens substituted by carbons), and R15Rneutral (a neutral Arg residue due to the a addition of a hydride in the zeta-carbon). A similar mechanism to the one used in our GSH activation proposal was implemented.

Glutathione transferase: new model for glutathione activation.

Glutathione transferases are enzymes of the cellular detoxification system that metabolize a vast spectrum of xenobiotic and endobiotic toxic compounds. They are homodimers or heterodimers and each monomer has an active center composed of a G-site in which glutathione (GSH) binds and an H-site for the electrophilic substrate. When GSH binds to the G-site, the pKa value of its thiol group drops by 2.5 units; this promotes its deprotonation and, therefore, produces a strong nucleophilic thiolate that is able to react with the electrophilic substrate. The mechanism behind the deprotonation of the thiol group is still unknown. Some studies point to the fact that the GSH glutamyl alpha-carboxylate group is essential for GSH activation, whereas others indicate the importance of the active-center water molecules. On the basis of QM/MM calculations, we propose a mechanism of GSH activation in which a water molecule, acting as a bridge, is able to assist in the transfer of the proton from the GSH thiol group to the GSH glutamyl alpha-carboxylate group, after an initial GSH conformational rearrangement. We calculated the potential of mean force of this GSH structural rearrangement that would be necessary for the approach of both groups and we then performed a QM/MM ONIOM scan of water-assisted proton transfer. The overall free-energy barrier for the process is consistent with experimental studies of the enzyme kinetics.

Mammalian cytosolic glutathione transferases.

Glutathione Transferases (GSTs) are crucial enzymes in the cell detoxification process catalyzing the nucleophilic attack of glutathione (GSH) on toxic electrophilic substrates and producing a less dangerous compound. GSTs studies are of great importance since they have been implicated in the development of drug resistance in tumoral cells and are related to human diseases such as Parkinson's, Alzheimer's, atherosclerois, liver cirrhosis, aging and cataract formation. In this review we start by providing an evolutionary perspective of the mammalian cytosolic GSTs known to date. Later on we focus on the more abundant classes alpha, mu and pi and their structure, catalysis, metabolic associated functions, drug resistance relation and inhibition methods. Finally, we introduce the recent insights on the GST class zeta from a metabolic perspective.