Positive Cooperative Binding Research Articles

The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases

Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced a positive cooperative binding phenomenon in this isoenzyme. Hence, intra-mitochondrial acidification, as occurring in astrocytes during glutamatergic transmission should favor the oxidative deamination of glutamate. Similar considerations apply to the handling of glutamate by the proximal convoluted tubules of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis.

Functional Analysis of Concatenated Heterotetrameric hERG1 Channels Reveals Requirement for Binding to Four Identical Sites to Achieve Full Activation by hERG1 Agonists

Recently, several small molecule activators of hERG1 K+ channels have been discovered, including PD-118057 (PD) and ICA-105574 (ICA). The putative binding sites for these two activators overlap and are located in a hydrophobic pocket between the S5 and S6 segments of adjacent hERG1 subunits. Thus, a homotetrameric hERG1 channel has potentially four identical binding sites. However, it is unclear whether drug binding to more than one site is required for full channel activation. Concatenated hERG1 tetramers containing wild-type and mutant subunits, heterologously expressed in Xenopus oocytes, were employed to determine the binding stoichiometry of hERG1 agonists. We previously found single mutation (L646E in S6 or F557L in S5) abolished the effects of PD and ICA, respectively. Concatenated tetramers were therefore constructed to contain a variable number (0 to 4) of the mutant subunits.PD increases hERG1 current by multiple effects, including a shift of inactivation to a more positive voltage, slowing of deactivation and an increase in single channel open probability. The efficacy of PD was increased in proportion to the WT:L646E subunit ratio. The Hill coefficient was 1.40 - 1.55 for all the heteromeric concatamers, suggesting a weak positive cooperativity for PD binding. At a fully effective concentration of PD (10μM), each WT binding site contributes ∼20% towards the maximum response of 80% enhancement of current.ICA increases hERG1 current by a profound attenuation of inactivation. An increase in the WT:F557L subunit ratio enhanced the agonist activity of ICA (10μM and 30μM) in a synergistic fashion. For 2WT/2F557L concatenated channels, synergy required adjacent (as opposed to diagonal) WT subunits. In summary, four intact binding sites per channel are required to fully activate hERG1 by PD or ICA.

Mechanism of Interaction between Bovine Serum Albumin and Sodium Dodecyl Sulfate

The binding isotherms of the interaction between bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS) were obtained using electromotive force measurements. Changes in the microenvironmental polarity of aromatic amino acid residues during the interaction were studied using fourth-derivative ultraviolet spectroscopy and fluorescence spectroscopy. The average number (ν) of SDS molecules bound to BSA increased with increasing SDS concentration. The polarity of tryptophan (Trp) residues decreased gradually and then remained almost constant. The polarity of tyrosine residues increased significantly and then decreased a little. The polarity of phenylalanine residues increased very slightly. The results show that SDS molecules bind to BSA in the vicinity of Trp-213 when ν gradually increases from 0 to 14. BSA unfolds from domain IIA, induced by SDS aggregates formed near Trp-213. The ν value then increases rapidly as a result of positive cooperative binding. When the ν value reaches about 302, saturation binding is achieved and the BSA conformation remains almost unchanged.

A 2.1-Å-Resolution Crystal Structure of Unliganded CRM1 Reveals the Mechanism of Autoinhibition

CRM1 mediates nuclear export of numerous proteins and ribonucleoproteins containing a leucine-rich nuclear export signal (NES). Binding of RanGTP to CRM1 in the nucleus stabilizes cargo association with CRM1, and vice versa, but the mechanism underlying the positive cooperativity in RanGTP and NES binding to CRM1 remains incompletely understood. Herein we report a 2.1-Å-resolution crystal structure of unliganded Saccharomyces cerevisiae CRM1 (Xpo1p) that demonstrates that an internal loop of CRM1 (referred to as HEAT9 loop) is primarily responsible for maintaining the NES-binding cleft in a closed conformation, rendering CRM1 incapable of NES binding in the absence of RanGTP. The structure also shows that the C-terminal tail of CRM1 stabilizes the autoinhibitory conformation of the HEAT9 loop and thereby reinforces autoinhibition. Comparison with the structures of CRM1-NES-RanGTP complexes reveals how binding of RanGTP is associated with a series of allosteric conformational changes in CRM1 that lead to opening of the NES-binding cleft, allowing for stable binding of NES cargoes.

X-linked Sideroblastic Anemia Due to Carboxyl-terminal ALAS2 Mutations That Cause Loss of Binding to the β-Subunit of Succinyl-CoA Synthetase (SUCLA2)

Mutations in the erythroid-specific aminolevulinic acid synthase gene (ALAS2) cause X-linked sideroblastic anemia (XLSA) by reducing mitochondrial enzymatic activity. Surprisingly, a patient with the classic XLSA phenotype had a novel exon 11 mutation encoding a recombinant enzyme (p.Met567Val) with normal activity, kinetics, and stability. Similarly, both an expressed adjacent XLSA mutation, p.Ser568Gly, and a mutation (p.Phe557Ter) lacking the 31 carboxyl-terminal residues also had normal or enhanced activity, kinetics, and stability. Because ALAS2 binds to the β subunit of succinyl-CoA synthetase (SUCLA2), the mutant proteins were tested for their ability to bind to this protein. Wild type ALAS2 bound strongly to a SUCLA2 affinity column, but the adjacent XLSA mutant enzymes and the truncated mutant did not bind. In contrast, vitamin B6-responsive XLSA mutations p.Arg452Cys and p.Arg452His, with normal in vitro enzyme activity and stability, did not interfere with binding to SUCLA2 but instead had loss of positive cooperativity for succinyl-CoA binding, an increased K(m) for succinyl-CoA, and reduced vitamin B6 affinity. Consistent with the association of SUCLA2 binding with in vivo ALAS2 activity, the p.Met567GlufsX2 mutant protein that causes X-linked protoporphyria bound strongly to SUCLA2, highlighting the probable role of an ALAS2-succinyl-CoA synthetase complex in the regulation of erythroid heme biosynthesis.

Cytochrome P450 3A4 Inhibition by Ketoconazole: Tackling the Problem of Ligand Cooperativity Using Molecular Dynamics Simulations and Free-Energy Calculations

Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of clinically used drugs and is often involved in adverse drug-drug interactions. It displays atypical binding and kinetic behavior toward a number of ligands characterized by a sigmoidal shape of the corresponding titration curves, which is indicative of a positive homotropic cooperativity. This requires a participation of at least two ligand molecules, whereby the binding of the first ligand molecule increases the affinity of CYP3A4 for the binding of the second ligand molecule. In the current study, a combination of molecular dynamics simulations and free-energy calculations was applied to elucidate the physicochemical origin of the observed positive homotropic cooperativity in ketoconazole binding to CYP3A4. The binding of the first ketoconazole molecule was established to increase the affinity for the binding of the second ketoconazole molecule by 5 kJ mol(-1), which explains and quantifies the experimentally observed cooperative behavior of CYP3A4. Shape complementarity through nonpolar van der Waals interactions was identified as the main driving force of this binding, which seems to be in line with the promiscuous nature of CYP3A4. Moreover, the calculated binding free energies were found to be in good agreement with the values predicted from a simple 2-ligand binding kinetic model as well as to successfully reproduce the experimental titration curve. This confirms the general applicability of rapid free-energy methods to study challenging biomolecular systems like cytochromes P450, which are characterized by a large flexibility and malleability of their active sites.

Structural Snapshots of Yeast Alkyl Hydroperoxide Reductase Ahp1 Peroxiredoxin Reveal a Novel Two-cysteine Mechanism of Electron Transfer to Eliminate Reactive Oxygen Species

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (C(P)) to Cys-62 and the resolving cysteine (C(R)) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of ∼2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 Å resolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing C(P) and C(R). An intermolecular C(P)-C(R) disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32.

Allosteric Modulator ORG27569 Induces CB1 Cannabinoid Receptor High Affinity Agonist Binding State, Receptor Internalization, and Gi Protein-independent ERK1/2 Kinase Activation

The cannabinoid receptor 1 (CB1), a member of the class A G protein-coupled receptor family, is expressed in brain tissue where agonist stimulation primarily activates the pertussis toxin-sensitive inhibitory G protein (G(i)). Ligands such as CP55940 ((1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3- hydroxypropyl)cyclohexan-1-ol) and Δ(9)-tetrahydrocannabinol are orthosteric agonists for the receptor, bind the conventional binding pocket, and trigger G(i)-mediated effects including inhibition of adenylate cyclase. ORG27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide) has been identified as an allosteric modulator that displays positive cooperativity for CP55940 binding to CB1 yet acts as an antagonist of G protein coupling. To examine this apparent conundrum, we used the wild-type CB1 and two mutants, T210A and T210I (D'Antona, A. M., Ahn, K. H., and Kendall, D. A. (2006) Biochemistry 45, 5606-5617), which collectively cover a spectrum of receptor states from inactive to partially active to more fully constitutively active. Using these receptors, we demonstrated that ORG27569 induces a CB1 receptor state that is characterized by enhanced agonist affinity and decreased inverse agonist affinity consistent with an active conformation. Also consistent with this conformation, the impact of ORG27569 binding was most dramatic on the inactive T210A receptor and less pronounced on the already active T210I receptor. Although ORG27569 antagonized CP55940-induced guanosine 5'-3-O-(thio)triphosphate binding, which is indicative of G protein coupling inhibition in a concentration-dependent manner, the ORG27569-induced conformational change of the CB1 receptor led to cellular internalization and downstream activation of ERK signaling, providing the first case of allosteric ligand-biased signaling via CB1. ORG27569-induced ERK phosphorylation persisted even after pertussis toxin treatment to abrogate G(i) and occurs in HEK293 and neuronal cells.

Thermodynamical study of interaction of histone H1 chromosomal protein and mitoxantrone anticancer drug

Using ultraviolet spectroscopy technique, we have investigated the interaction of anticancer drug, mitoxantrone with calf thymus histone H1 chromosomal protein in 100 mM phosphate buffer, pH 7.0, at temperatures 300 and 310 K. UV spectroscopy results show interactions between mitoxantrone and histone H1 with a positive cooperative binding process which was confirmed by Scatchard plot. According to the obtained results, it is concluded that histone H1 can be considered as a target for mitoxantrone binding at the chromatin level.

Internal Motions and Exchange Processes in Human Ileal Bile Acid Binding Protein As Studied by Backbone 15N Nuclear Magnetic Resonance Spectroscopy

Human ileal bile acid binding protein (I-BABP), a member of the family of intracellular lipid binding proteins, is thought to play a role in the enterohepatic circulation of bile salts. Previously, we have shown by stopped-flow fluorescence analysis that positive binding cooperativity exhibited by I-BABP in its interactions with glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two primary bile salts in humans, is related to a slow conformational change in the protein. In this study, we used backbone (15)N relaxation nuclear magnetic resonance (NMR) techniques to obtain residue-specific information about the internal dynamics of apo I-BABP and the doubly ligated I-BABP:GCA:GCDA complex on various time scales. According to our NMR data, bile salt binding is accompanied by a slight rigidification of the (15)N-(1)H bond vectors on the picosecond to nanosecond time scale, with most pronounced changes occurring in the C-D region. In contrast to the minor effects of ligation on fast motions, relaxation dispersion NMR experiments indicate a marked difference between the two protein states on the microsecond to millisecond time scale. In the apo form, an extensive network of conformational fluctuations is detected throughout segments of the EFGHIJ β-strands and the C-D loop, which cease upon complexation. Our NMR data are in agreement with a conformational selection model we proposed earlier for I-BABP and support the hypothesis of an allosteric mechanism of ligand binding. According to the NMR measurements, the helical cap region may have a less crucial role in mediating ligand entry and release than what has been indicated for fatty acid binding proteins.

Eliminating ATP Binding in Specific ClpX Subunits Yields Functional ATP-Fueled Protein-Unfolding Machines

AAA+ ring hexamers of ClpX utilize the energy of ATP binding and hydrolysis to unfold protein substrates and translocate the resulting denatured polypeptide into the degradation chamber of ClpP, an associated self-compartmentalized peptidase. Nucleotide-dependent conformational changes are necessary for ClpX binding to ClpP and to protein substrates as well as for allosteric machine function. ClpX functions asymmetrically. In crystal structures, for example, four loadable subunits are competent for nucleotide binding, whereas two unloadable subunits are not. Moreover, ATP hydrolysis in one subunit can power conformational changes in the entire ring and protein degradation. Subunit-subunit interactions are essential for ClpX machine function, but the communication mechanisms are poorly defined. For instance, it is not known whether the conformations of loadable and unloadable subunits remain fixed or interchange during the hundreds of cycles of ATP hydrolysis that are required for protein unfolding, translocation, and degradation. To probe subunit communication, we constructed covalently linked mutant hexamers in which the nucleotide affinity of specific subunits was dramatically reduced by mutations in the Walker A, sensor-II, or box II motifs and developed novel fluorescence assays to probe nucleotide-binding cooperativity as well as ATP binding to specific subunits. Strikingly, ClpX pseudo-hexamers bearing two opposed subunits with severe ATP-binding defects hydrolyze ATP at near normal rates and are able to unfold and translocate protein substrates. For some variants, machine function was retained despite sensor-II-dependent abrogation of the positive cooperativity of ATP binding. Because hexamers with two “permanent” unloadable subunits retain basic ClpX machine functionality, subunit switching between unloadable and loadable conformations does not appear to be required for protein unfolding or translocation. Our results are most consistent with probabilistic models of ATP binding and hydrolysis rather than strictly sequential models.

Conversion of Molecular Information by Luminescent Nanointerface Self-Assembled from Amphiphilic Tb(III) Complexes

A novel amphiphilic Tb(3+) complex (TbL(+)) having anionic bis(pyridine) arms and a hydrophobic alkyl chain is developed. It spontaneously self-assembles in water and gives stable vesicles that show sensitized luminescence of Tb(3+) ions at neutral pH. This TbL(+) complex is designed to show coordinative unsaturation, i.e., water molecules occupy some of the first coordination spheres and are replaceable upon binding of phosphate ions. These features render TbL(+) self-assembling receptor molecules which show increase in the luminescence intensity upon binding of nucleotides. Upon addition of adenosine triphosphate (ATP), significant amplification of luminescent intensity was observed. On the other hand, ADP showed moderately increased luminescence and almost no enhancement was observed for AMP. Very interestingly, the increase in luminescence intensity observed for ATP and ADP showed sigmoidal dependence on the concentration of added nucleotides. It indicates positive cooperative binding of these nucleotides to TbL(+) complexes preorganized on the vesicle surface. Self-assembly of amphiphilic Tb(3+) receptor complexes provides nanointerfaces which selectively convert and amplify molecular information of high energy phosphates linked by phosphoanhydride bonds into luminescence intensity changes.

Positive and negative cooperativity of agonist and allosteric modulator binding in alpha7 nAChR: Looking for the therapeutic window

Positive and negative cooperativity of agonist and allosteric modulator binding in alpha7 nAChR: Looking for the therapeutic window

Positive cooperative mechanistic binding of proteins at low concentrations: A comparison of poly (sodium N-undecanoyl sulfate) and sodium dodecyl sulfate

The interactions of the negatively charged achiral molecular micelle, poly (sodium N-undecanoyl sulfate) (poly-SUS), with four different proteins using intrinsic and extrinsic fluorescence spectroscopic probes, are studied. A comparison of poly-SUS with the conventional surfactant, sodium dodecyl sulfate (SDS), and the monomeric species, SUS, is also reported. In this work, we observed that poly-SUS preferentially binds to acidic proteins, exhibiting positive cooperativity at concentrations less than 1mM for all proteins studied. Moreover, it appears that the hydrophobic microdomain formed through polymerization of the terminal vinyl group of the monomer, SUS, is largely responsible for the superior binding capacity of poly-SUS. From these results, we conclude that the interactions of poly-SUS with the acidic proteins are predominantly hydrophobic and postulate that poly-SUS would produce superior interactions relative to SDS at low concentrations in polyacrylamide gel electrophoresis (PAGE). As predicted, use of poly-SUS allowed separation of the His-tagged tumor suppressor protein, p53, at sample buffer concentrations as low as 0.08% w/v (2.9mM), which is 24 times lower than required for SDS in the standard reducing PAGE protocol. This work highlights the use of poly-SUS as an effective surfactant in 1D biochemical analysis.

NMR structures of apo L. casei dihydrofolate reductase and its complexes with trimethoprim and NADPH: contributions to positive cooperative binding from ligand-induced refolding, conformational changes, and interligand hydrophobic interactions.

In order to examine the origins of the large positive cooperativity (ΔG0coop = −2.9 kcal mol−1) of trimethoprim (TMP) binding to a bacterial dihydrofolate reductase (DHFR) in the presence of NADPH, we have determined and compared NMR solution structures of L. casei apo DHFR and its binary and ternary complexes with TMP and NADPH and made complementary thermodynamic measurements. The DHFR structures are generally very similar except for the A−B loop region and part of helix B (residues 15−31) which could not be directly detected for L. casei apo DHFR because of line broadening from exchange between folded and unfolded forms. Thermodynamic and NMR measurements suggested that a significant contribution to the cooperativity comes from refolding of apo DHFR on binding the first ligand (up to −0.95 kcals mol−1 if 80% of A−B loop requires refolding). Comparisons of Cα−Cα distance differences and domain rotation angles between apo DHFR and its complexes indicated that generally similar conformational changes involving domain movements accompany formation of the binary complexes with either TMP or NADPH and that the binary structures are approaching that of the ternary complex as would be expected for positive cooperativity. These favorable ligand-induced structural changes upon binding the first ligand will also contribute significantly to the cooperative binding. A further substantial contribution to cooperative binding results from the proximity of the bound ligands in the ternary complex: this reduces the solvent accessible area of the ligand and provides a favorable entropic hydrophobic contribution (up to −1.4 kcal mol−1).

Mass Spectrometry Reveals That the Antibiotic Simocyclinone D8 Binds to DNA Gyrase in a “Bent-Over” Conformation: Evidence of Positive Cooperativity in Binding

DNA topoisomerases are enzymes that control DNA topology and are vital targets for antimicrobial and anticancer drugs. Here we present a mass spectrometry study of complexes formed between the A subunit of the topoisomerase DNA gyrase and the bifunctional inhibitor simocyclinone D8 (SD8), an antibiotic isolated from Streptomyces. These studies show that, in an alternative mode of interaction to that found by X-ray crystallography, each subunit binds a single bifunctional inhibitor with separate binding pockets for the two ends of SD8. The gyrase subunits form constitutive dimers, and fractional occupancies of inhibitor-bound states show that there is strong allosteric cooperativity in the binding of two bifunctional ligands to the dimer. We show that the mass spectrometry data can be fitted to a general model of cooperative binding via an extension of the "tight-binding" approach, providing a rigorous determination of the dissociation constants and degree of cooperativity. This general approach will be applicable to other systems with multiple binding sites and highlights mass spectrometry's role as a powerful emerging tool for unraveling the complexities of biomolecular interactions.

Polyoxyethylene alkyl and nonyl phenol ethers complexation with potato starch

Starchy soil removal efficiency when cleaning food industry equipment can be affected by surfactant–starch complexes. Polyoxyethylene alkyl and nonyl phenol ethers complexation with potato starch has been studied by surface tension measurements. All surfactants assayed yield inclusion complexes with starch after a critical concentration of association was achieved. No starch saturation was observed throughout complexation until micellization, with bound surfactant being proportional to the total added surfactant. This bound/total surfactant ratio and the critical concentration of association seemed to depend mainly on the surfactant alkyl chain length. Unlike fatty alcohol ethoxylates, the nonyl phenol ethoxylate was feebly bound likely caused by its aromatic moiety. Binding isotherms and Scatchard plots suggested surfactants positive cooperative and non-cooperative binding with amylose and amylopectin respectively. Phosphate buffer raised surfactants binding capacity, ascribed either to an increase in cooperativity or to a reduction in the number of glucose units required to bind a surfactant molecule.

Activation of PKR by Stem Loop RNAs Requires Flanking ssRNA Arms

Protein kinase R (PKR) is a key component of the interferon-induced viral response pathway. PKR contains an N-terminal double-stranded RNA (dsRNA) binding domain and a C-terminal kinase domain connected by a ∼90-residue linker. Upon binding dsRNA, PKR undergoes autophosphorylation and kinase activation. Activated PKR then phosphorylates the alpha subunit of the eukaryotic initiation growth factor 2 and thus inhibits protein synthesis in virally infected cells. A minimum of 30 bp of dsRNA is required to bind two PKRs, leading to dimerization and activation. Stem-loops are common RNA structural motifs. Recently, it was reported that short stem-loops containing 16 bp and ssRNA flanking arms are also able to activate PKR and that activation requires a 5’-triphosphate. However, the activation mechanism is not known. We have prepared RNAs containing a 15 bp stem-loop and various ssRNA flanking arms and characterized activation and binding of PKR. Activation by these RNAs requires ssRNA arms on both the 5’ and 3’ sides but it is independent of the presence of a 5’-triphosphate. Sedimentation velocity measurements indicate that a single PKR binds to the activating stem-loop RNA at 200 mM NaCl with Kd = 309 nM. PKR binding affinity is not strongly affected by removal of either of the ssRNA arms or the 5’-triphosphate. At lower [NaCl] (75 mM), each of the stem-loop RNAs can bind two monomers of PKR. However, only the activating RNA exhibits a positive binding cooperativity. We propose that activation by this RNA is due to enhanced population of the species containing two bound PKRs as a result of cooperative binding of the second PKR monomer.

A β-to-β 2,5-thienylene-bridged cyclic porphyrin tetramer: its rational synthesis and 1 : 2 binding mode with C60

A β-to-β2,5-thienylene-bridged cyclic porphyrin tetramer was rationally synthesized via a concise synthetic route. The tetraporphyrin exhibits a positive cooperative binding ability to C60 and demonstrates a new potential of the nonplanar, distorted cyclic porphyrin arrays.

Thermodynamic and NMR analysis of inhibitor binding to dihydrofolate reductase

Isothermal titration calorimetry (ITC) was used to determine the thermodynamic driving force for inhibitor binding to the enzyme dihydrofolate reductase (DHFR) from Escherichia coli. 1,4-Bis-{[ N-(1-imino-1-guanidino-methyl)]sulfanylmethyl}-3,6-dimethyl-benzene ( 1) binds DHFR:NADPH with a K d of 13 ± 5 nM while the related inhibitor 1-{[ N-(1-imino-guanidino-methyl)]sulfanylmethyl}-3-trifluoromethyl-benzene ( 2) binds DHFR:NADPH with a K d of 3.2 ± 2.2 μM. The binding of these inhibitors has both a favorable entropy and enthalpy of binding. Additionally, we observe positive binding cooperativity between both 1 and 2 and the cofactor NADPH. Binding of compound 1 to DHFR is 285-fold tighter in the presence of the NADPH cofactor than in its absence. We did not detect binding of 2 to DHFR in the absence of NADPH. The backbone amide 1H and 15N NMR resonances of DHFR:NADPH and both DHFR:NADPH inhibitor complexes were assigned in order to better understand the binding of these inhibitors in solution. The chemical shift perturbations observed with the binding of 1 were greatest at residues closest to the binding site, but significant perturbations also occur away from the inhibitor location at amino acids in the vicinity of residue 58 and in the GH loop. The pattern of chemical shift changes observed with the binding of 2 is similar to that seen with 1. The main differences in chemical shift perturbation between the two inhibitors are in the Met20 loop and in residues at the interface between the inhibitor and NADPH.