Dihydrofolate Reductase In Complex Research Articles

Perturbative diffraction methods resolve a conformational switch that facilitates a two-step enzymatic mechanism

Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident with Escherichia coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps.

A computational investigation of potential plant-based bioactive compounds against drug-resistant Staphylococcus aureus of multiple target proteins

Drug-resistant Staphylococcus aureus (DRSA) poses a significant global health threat, like bacteremia, endocarditis, skin, soft tissue, bone, and joint infections. Nowadays, the resistance against conventional drugs has been a prompt and focused medical concern. The present study aimed to explore the inhibitory potential of plant-based bioactive compounds (PBBCs) against effective target proteins using a computational approach. We retrieved and verified 22 target proteins associated with DRSA and conducted a screening process that involved testing 87 PBBCs. Molecular docking was performed between screened PBBCs and reference drugs with selected target proteins via AutoDock. Subsequently, we filtered the target proteins and top PBBCs based on their binding affinity scores. Furthermore, molecular dynamic simulation was carried out through GROMACS for a duration of 100 ns, and the binding free energy was calculated using the gmx_MMPBSA. The result showed consistent hydrogen bonding interactions among the amino acid residues Ser 149, Arg 151, Thr 165, Thr 216, Glu 239, Ser 240, Ile 14, as well as Asn 18, Gln 19, Lys 45, Thr 46, Tyr 109, with their respective target proteins of the penicillin-binding protein and dihydrofolate reductase complex. Additionally, we assessed the pharmacokinetic properties of screened PBBCs via SwissADME and AdmetSAR. The findings suggest that β-amyrin, oleanolic acid, kaempferol, quercetin, and friedelin have the potential to inhibit the selected target proteins. In future research, both in vitro and in vivo, experiments will be needed to establish these PBBCs as potent antimicrobial drugs for DRSA. Communicated by Ramaswamy H. Sarma.

The Complementarity Principle-One More Step towards Analytical Docking on the Example of Dihydrofolate Reductase Complexes.

New approaches to assessing the “enzyme–ligand” complementarity, taking into account hydrogens, have been proposed. The approaches are based on the calculation of three-dimensional maps of the electron density of the receptor–ligand complexes. The action of complementarity factors, first proposed in this article, has been demonstrated on complexes of human dihydrofolate reductase (DHFR) with ligands. We found that high complementarity is ensured by the formation of the most effective intermolecular contacts, which are provided due to predominantly paired atomic–atomic interactions, while interactions of the bifurcate and more disoriented type are minimized. An analytical docking algorithm based on the proposed receptor–ligand complementarity factors is proposed.

Electronic properties investigation of human dihydrofolate reductase complexes with ligands

Despite the fact that there are already drugs for cancer, they still show strong toxicity to the human organism. That is why it is necessary to establish the factors affecting activity in order to develop new, more effective drugs aimed at tumor cells, minimizing harm to healthy cells. The present research is based on electronic properties calculation of the complexes using AlteQ approach. In the focus of this study are complexes of human dihydrofolate reductase (hDHFR) with a series of known inhibitors bound in the active site. Further, a statistical analysis was performed to establish the relationships between a myriad electronic characteristics and IC50. The change in total volume and the change of own electrons number of hydrogen atoms in their atomic basins are identified as the descriptors correlating the most with the hDHFR inhibition potency. Additionally, two lipophilic parts of protein (Thr56, Ser59, Ile60 and Ile7, Val8, Ala9) were found, which act as a key factor in decreasing bioactivity. The depth analysis of intermolecular interactions showed that the interactions between water molecules and ligand play a crucial role in hDHFR inhibition. Furthermore, the molecular dynamics simulations were used for deeper understanding of the structural inhibition, each for 50 ns time scale in explicit water conditions. Thus, the AlteQ approach made it possible to determine the factors influencing the activity and evaluate them not only qualitatively, but also quantitatively. Communicated by Ramaswamy H. Sarma

Crystal structures of the closed form of Mycobacterium tuberculosis dihydrofolate reductase in complex with dihydrofolate and antifolates.

Tuberculosis is a disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious pathogen, with a high prevalence in developing countries in Africa and Asia. There still is a need for the development or repurposing of novel therapies to combat this disease owing to the long-term nature of current therapies and because of the number of reported resistant strains. Here, structures of dihydrofolate reductase from M.tuberculosis (MtDHFR), which is a key target of the folate pathway, are reported in complex with four antifolates, pyrimethamine, cycloguanil, diaverdine and pemetrexed, and its substrate dihydrofolate in order to understand their binding modes. The structures of all of these complexes were obtained in the closed-conformation state of the enzyme and a fine structural analysis indicated motion in key regions of the substrate-binding site and different binding modes of the ligands. In addition, the affinities, through Kd measurement, of diaverdine and methotrexate have been determined; MtDHFR has a lower affinity (highest Kd) for diaverdine than pyrimethamine and trimethoprim, and a very high affinity for methotrexate, as expected. The structural comparisons and analysis described in this work provide new information about the plasticity of MtDHFR and the binding effects of different antifolates.

Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase.

Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein-ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not.

Structural Characterization of Dihydrofolate Reductase Complexes by Top-Down Ultraviolet Photodissociation Mass Spectrometry.

The stepwise reduction of dihydrofolate to tetrahydrofolate entails significant conformational changes of dihydrofolate reductase (DHFR). Binary and ternary complexes of DHFR containing cofactor NADPH, inhibitor methotrexate (MTX), or both NADPH and MTX were characterized by 193 nm ultraviolet photodissociation (UVPD) mass spectrometry. UVPD yielded over 80% sequence coverage of DHFR and resulted in production of fragment ions that revealed the interactions between DHFR and each ligand. UVPD of the binary DHFR·NADPH and DHFR·MTX complexes led to an unprecedented number of fragment ions containing either an N- or C-terminal protein fragment still bound to the ligand via retention of noncovalent interactions. In addition, holo-fragments retaining both ligands were observed upon UVPD of the ternary DHFR·NADPH·MTX complex. The combination of extensive holo and apo fragment ions allowed the locations of the NADPH and MTX ligands to be mapped, with NADPH associated with the adenosine binding domain of DHFR and MTX interacting with the loop domain. These findings are consistent with previous crystallographic evidence. Comparison of the backbone cleavage propensities for apo DHFR and its holo counterparts revealed significant variations in UVPD fragmentation in the regions expected to experience conformational changes upon binding NADPH, MTX, or both ligands. In particular, the subdomain rotation and loop movements, which are believed to occur upon formation of the transition state of the ternary complex, are reflected in the UVPD mass spectra. The UVPD spectra indicate enhanced backbone cleavages in regions that become more flexible or show suppressed backbone cleavages for those regions either shielded by the ligand or involved in new intramolecular interactions. This study corroborates the versatility of 193 nm UVPD mass spectrometry as a sensitive technique to track enzymatic cycles that involve conformational rearrangements.

Quartz crystal microbalance with dissipation and microscale thermophoresis as tools for investigation of protein complex formation between thymidylate synthesis cycle enzymes

Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) play essential role in DNA synthesis, repair and cell division by catalyzing two subsequent reactions in thymidylate biosynthesis cycle. The lack of either enzyme leads to thymineless death of the cell, therefore inhibition of the enzyme activity is a common and successful tool in cancer chemotherapy and treatment of other diseases. However, the detailed mechanism of thymidylate synthesis cycle, especially the interactions between cycle enzymes and its role remain unknown.In this paper we are the first to show that human TS and DHFR enzymes form a strong complex which might be essential for DNA synthesis. Using two unique biosensor techniques, both highly sensitive to biomolecular interactions, namely quartz crystal microbalance with dissipation monitoring (QCM-D) and microscale thermophoresis (MST) we have been able to determine DHFR–TS binding kinetic parameters such as the Kd value being below 10µM (both methods), kon=0.46×104M−1s−1 and koff=0.024s−1 (QCM-D). We also calculated Gibbs free energy as in the order of −30kJ/mol and DHFR/TS molar ratio pointing to binding of 6 DHFR monomers per 1 TS dimer (both methods). Moreover, our data from MST analysis have pointed to positive binding cooperativity in TS–DHFR complex formation. The results obtained with both methods are comparable and complementary.

Crystal Cryocooling Distorts Conformational Heterogeneity in a Model Michaelis Complex of DHFR

Most macromolecular X-ray structures are determined from cryocooled crystals, but it is unclear whether cryocooling distorts functionally relevant flexibility. Here we compare independently acquired pairs of high-resolution data sets of a model Michaelis complex of dihydrofolate reductase (DHFR), collected by separate groups at both room and cryogenic temperatures. These data sets allow us to isolate the differences between experimental procedures and between temperatures. Our analyses of multiconformer models and time-averaged ensembles suggest that cryocooling suppresses and otherwise modifies side-chain and main-chain conformational heterogeneity, quenching dynamic contact networks. Despite some idiosyncratic differences, most changes from room temperature to cryogenic temperature are conserved and likely reflect temperature-dependent solvent remodeling. Both cryogenic data sets point to additional conformations not evident in the corresponding room temperature data sets, suggesting that cryocooling does not merely trap preexisting conformational heterogeneity. Our results demonstrate that crystal cryocooling consistently distorts the energy landscape of DHFR, a paragon for understanding functional protein dynamics.

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10Å from the active site, while epitope β is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a β epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.

Side-Chain Conformational Heterogeneity of Intermediates in the Escherichia coli Dihydrofolate Reductase Catalytic Cycle

Escherichia coli dihydrofolate reductase (DHFR) provides a paradigm for the integrated study of the role of protein dynamics in enzyme function. Previous studies of backbone and side chain dynamics have yielded unprecedented insights into the mechanism by which DHFR progresses through the structural changes that occur during its catalytic cycle. Here we report a comprehensive study of the χ1 rotamer populations of the aromatic and γ-methyl containing residues for complexes of the catalytic cycle, based on NMR measurement of (3)JCγCO and (3)JCγN coupling constants. We report conformational and dynamic information for eight distinct complexes, where transitions between rotamer wells may occur on a broad picosecond to millisecond time scale. This large volume of (3)J data has allowed us to fit new Karplus parameterizations for aromatic side chains and to select the best available of previously determined parameters for Ile, Thr, and Val. The (3)JCγCO and (3)JCγN coupling constants are found to be extremely sensitive measures of side chain χ1 rotamers and to give important insights into the extent of conformational averaging. For a subset of residues in DHFR, the extent of rotamer averaging is invariant to the nature of the bound ligand, while for other residues the rotamer averaging differs in one or more complexes of the enzymatic cycle. These variable-averaging residues are generally located near the active site, but the phenomenon extends into the adenosine binding domain. For several residues, the rotamer populations in different DHFR complexes appear to depend on whether the complex is in the closed or occluded state, and some residues are exquisitely sensitive to small changes in the nature of the bound ligand.

Identification of endogenous ligands bound to bacterially expressed human and E. coli dihydrofolate reductase by 2D NMR

Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and Escherichia coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ecDHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed “humanized” mutant of ecDHFR switches binding specificity in the cell.

Structural analysis of human dihydrofolate reductase as a binary complex with the potent and selective inhibitor 2,4-diamino-6-{2'-O-(3-carboxypropyl)oxydibenz[b,f]-azepin-5-yl}methylpteridine reveals an unusual binding mode.

In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structure of the binary complex of human dihydrofolate reductase (hDHFR) with the potent and selective inhibitor 2,4-diamino-6-{2'-O-(3-carboxypropyl)oxydibenz[b,f]-azepin-5-yl}methylpteridine (PT684) was determined to 1.8 Å resolution. These data revealed that the carboxylate side chain of PT684 occupies two alternate positions, neither of which interacts with the conserved Arg70 in the active-site pocket, which in turn hydrogen bonds to water. These observations are in contrast to those reported for the ternary complex of mouse DHFR (mDHFR) with NADPH [Cody et al. (2008), Acta Cryst. D64, 977-984], in which the 3-carboxypropyl side chain of PT684 was hydrolyzed to its hydroxyl derivative, PT684a. The crystallization conditions differed for the human and mouse DHFR crystals (100 mM K2HPO4 pH 6.9, 30% ammonium sulfate for hDHFR; 15 mM Tris pH 8.3, 75 mM sodium cacodylate, PEG 4K for mDHFR). Additionally, the side chains of Phe31 and Gln35 in the hDHFR complex have a single conformation, whereas in the mDHFR complex they occupied two alternative conformations. These data show that the hDHFR complex has a decreased active-site volume compared with the mDHFR complex, as reflected in a relative shift of helix C (residues 59-64) of 1.2 Å, and a shift of 1.5 Å compared with the ternary complex of Pneumocystis carinii DHFR (pcDHFR) with the parent dibenz[b,f]azepine PT653. These data suggest that the greater inhibitory potency of PT684 against pcDHFR is consistent with the larger active-site volume of pcDHFR and the predicted interactions of the carboxylate side chain with Arg75.

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry for Probing the Microenvironment of Histidine Residues in Dihydrofolate Reductase

BackgroundHistidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C2-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.Methodology/Principal FindingsUsing His-HDX-MS, the pK a values and the half-lives (t 1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK a and t 1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK a, t 1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK a and t 1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.Conclusions/SignificanceOur results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

Role of Mutations in Dihydrofolate Reductase DfrA (Rv2763c) and Thymidylate Synthase ThyA (Rv2764c) in Mycobacterium tuberculosis Drug Resistance

We would like to comment on a number of recent reports in this journal (6, 8, 12, 18) concerning Mycobacterium tuberculosis dihydrofolate reductase (DHFR), encoded by dfrA (Rv2763c). Around 36% of phenotypically para-aminosalicylic acid (PAS)-resistant M. tuberculosis strains harbor mutations in thyA (Rv2764c), which encodes a thymidylate synthase (20). In their effort to elucidate the remaining unknown resistance mechanism(s), Mathys et al. extended their sequence analysis to a number of additional genes, including dfrA (12). It was unclear whether the three dfrA mutations they identified in the PAS-resistant strains P-693 and P-3158 could contribute to PAS resistance on their own. Nonetheless, these findings are notable for two reasons. First, isoniazid (INH) has been shown to inhibit M. tuberculosis DHFR in vitro (1). Whether the same holds true for ethionamide, which shares a number of common resistance mechanisms with INH, was not tested (J. Blanchard, personal communication). In any case, the clinical relevance of DHFR-mediated INH resistance remains enigmatic. To date, only Ho et al. have addressed this question, but they did not identify any dfrA mutations in a screen of 127 INH-resistant clinical isolates (8). Consequently, Mathys et al. remain the first to describe mutations in this target (12). However, given that isolates with mutated DHFR are members of a cluster with baseline INH resistance, the importance of these mutations with respect to INH resistance remains unclear. Irrespective of their relevance in INH resistance, these dfrA mutations are noteworthy for a second reason. Contrary to previous wisdom, Forgacs et al. recently showed that M. tuberculosis is sensitive to the drug combination trimethoprim-sulfamethoxazole (TMP-SMX) (6, 18). DHFR is competitively inhibited by TMP, and consequently, mutations therein lead to resistance in a variety of organisms (9, 16, 19). The crystal structures of the wild-type M. tuberculosis DHFR in complex with NADPH alone and with NADPH combined with the inhibitor TMP, methotrexate, or an analogue of the antimalarial agent WR99210 have been solved (11). Interestingly, the wild-type 66Ser, which is mutated to Cys in strain P-693 (12), interacts directly with NADPH via an H bond and a hydrophobic interaction. Similarly, the wild-type 54Val, which is mutated to Ala in strain P-3158 (in addition to a second 110Cys→Arg change), has a hydrophobic interaction with BR-WR99210 (11). Should these amino acid substitutions alter NADPH interactions or inhibitor binding, this raises the possibility that these PAS-resistant strains might also be TMP-SMX resistant. To investigate this possibility, phenotypic susceptibility testing of both isolates, as described by Forgacs et al., is warranted (6, 12). In addition, the treatment history of both patients should be reviewed for evidence of treatment with TMP-SMX. We would now like to turn to the mutations in M. tuberculosis thyA, which were the focus of a number of studies (12, 13, 20). We note that the mutational loss of ThyA leads to TMP resistance in other organisms. Under these circumstances, tetrahydrofolate, the product of DHFR, is no longer required to regenerate N5,N10-methylenetetrahydrofolate, which would otherwise be oxidized by ThyA in the deoxyuridylate methylation process. Consequently, TMP-mediated competitive inhibition of DHFR can be tolerated by the cell (10, 13). In the case of Staphylococcus aureus, thyA mutations are also responsible for a small-colony-variant phenotype and are associated with hypermutability, which is thought to favor adaptation to long-term persistence and further antibiotic resistance (3-5, 14). Taken together, this raises the possibility that thyA mutations that account for PAS resistance in M. tuberculosis might also lead to cross-resistance to TMP-SMX (6, 18). In fact, in their effort to show the importance of thyA mutations for PAS resistance in M. tuberculosis, Rengarajan et al. showed that transposon-disrupted thyA from Mycobacterium bovis BCG was not able to rescue a ThyA− phenotype in Escherichia coli and restore TMP sensitivity (13). Moreover, they showed that these PAS-resistant M. bovis BCG strains harboring a transposon in thyA were resistant to the DHFR inhibitor 8710, which, unlike TMP on its own, is active against wild-type M. bovis BCG (13, 15). Should this hold true for TMP-SMX, this could have obvious implications for the utility of these antibiotics, comparable to the situation of isoniazid and ethionamide (17). To assess the possible clinical impact and the degree of cross-resistance, we must quantify the proportion of laboratory and clinical TMP-SMX resistance which might be due to thyA mutations rather than mutation in DHFR or due to a third resistance mechanism involving the dihydropteroate synthase FolP1 (Rv3608c) (2, 7, 10). This also has practical implications for the experiment we suggested above. Mathys et al. should include P strains from their collection that carry the same double thyA mutations as P-693 and P-3158 but have a wild-type DHFR as the control (12). We hope that these observations will spur a thorough preclinical examination of TMP-SMX, as called for by Young (18).

The Z isomer of 2,4-diaminofuro[2,3-d]pyrimidine antifolate promotes unusual crystal packing in a human dihydrofolate reductase ternary complex.

The crystal structure of the ternary complex of human dihydrofolate reductase (hDHFR) with NADPH and the Z isomer of 2,4-diamino-5-[2-(2'-methoxyphenyl)propenyl]-furo[2,3-d]pyrimidine (Z1) shows that the Z isomer binds in the normal antifolate orientation in which the furo oxygen occupies the 8-amino position observed in the binding of 2,4-diaminopteridine antifolates such as methotrexate and with the methoxyphenyl moiety cis to and coplanar with the furo[2,3-d]pyrimidine ring. The hDHFR ternary complex crystallized in the orthorhombic space group P2(1)2(1)2(1) and its structure was refined to 1.7 A resolution. Although other hDHFR complexes crystallize in this space group, these data provide only the second example of an unusual packing arrangement in which the conserved active-site Arg70 forms a salt bridge to the side chain of Glu44 from a symmetry-related molecule. As a result, the conformations of Phe31 and Gln35 shift with respect to those observed in the structure of mouse DHFR bound to Z1, which crystallizes in the monoclinic space group P2(1) and shows that Gln35 interacts with Arg70.

Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State

The arduous task of rationally designing small-molecule enzyme inhibitors is complicated by the inherent flexibility of the protein scaffold. To gain insight into the changes in dynamics associated with small-molecule-based inhibition, we have characterized, using NMR spectroscopy, Escherichia coli dihydrofolate reductase in complex with two drugs: methotrexate and trimethoprim. The complexes allowed the intrinsic dynamic effects of drug binding to be revealed within the context of the "closed" structural ensemble. Binding of both drugs results in an identical decoupling of global motion on the micro- to millisecond timescale. Consistent with a change in overall dynamic character, the drugs' perturbations to pico- to nanosecond backbone and side-chain methyl dynamics are also highly similar. These data show that the inhibitors simultaneously modulate slow concerted switching and fast motions at distal regions of dihydrofolate reductase, providing a dynamic link between the substrate binding site and distal loop residues known to affect catalysis.

Bis[2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidin-1-ium]DL-malate

Racemic malic acid and trimethoprim [5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine] form a 1:2 salt (monoclinic, P2(1)/c), 2C(14)H(19)N(4)O(3)(+).C(4)H(4)O(5)(2-), in which the malate component is disordered across a centre of inversion. The crystal structure of the salt consists of protonated trimethoprim residues and a malate dianion. The carboxylate group of the malate ion interacts with the trimethoprim cation in a linear fashion through pairs of N-H...O hydrogen bonds to form a cyclic hydrogen-bonded motif. This is similar to the carboxylate-trimethoprim cation interaction observed earlier in the complex of dihydrofolate reductase with trimethoprim. The structure of the salt of trimethoprim with racemic DL-malic acid reported here is the first of its kind. The present study investigates the conformations and the hydrogen-bonding interactions, which are very important for biological functions. The pyrimidine plane makes a dihedral angle of 78.08 (7) degrees with the benzene ring of the trimethoprim cation. The cyclic hydrogen-bonded motif observed in this structure is self-organized, leading to novel types of hydrogen-bonding motifs in supramolecular patterns.

Structural analysis of a holoenzyme complex of mouse dihydrofolate reductase with NADPH and a ternary complex with the potent and selective inhibitor 2,4-diamino-6-(2'-hydroxydibenz[b,f]azepin-5-yl)methylpteridine.

It has been shown that 2,4-diamino-6-arylmethylpteridines and 2,4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2'-hydroxydibenz[b,f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 A resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed no electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2'-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59-64) by 0.6 A compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.

Development of a chemical screening system using aqueorin-fused protein

We developed a unique screening system that consists of combination of high photo-sensitivity of photoprotein aequorin (AQ) and our developed high-performance affinity purification system. In the present study, we demonstrated to detect the specific interaction between methotrexate (MTX) and its target dihydrofolate reductase (DHFR) fused with AQ. We succeeded to prepare highly purified AQ-fused DHFR, which showed high sensitive light emission. To test the screening system, we prepared the complex of MTX-immobilized magnetic nanobeads and AQ-fused DHFR. Bound AQ-fused DHFR with the beads was specifically released by addition of MTX. Thus, this methodology enables us to search a novel chemical that binds to target proteins without complicated processes. Furthermore, thank to the highly sensitive luminescence intensity of AQ, this methodology would be performed in very small scale with high responsibility, leading to development of high throughput screening systems.