Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase

Invariant arginine 179, one of four arginines that are conservedin all thymidylate synthases (TS) and that bind the phosphatemoiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP),can be altered even to a negatively charged glutainic acid withlittle effect on k_cat. In the mutant structures, ordered wateror the other phosphate binding arginines compensate for thehydrogen bonds made by Arg179 in the wild-type enzyme and thereis almost no change in the conformation or binding site of dUMP.Correlation of dUMP K_ds for TS R179A and TS R179K with the structuresof their binary complexes shows that the positive charge onArg179 contributes significantly to dUMP binding affinity. k_cat/Kmfor dUMP measures the rate of dUMP binding to TS during theordered bi-substrate reaction, and in the ternary complex dUMPprovides a binding surface for the cofactor. k_cat/Km reflectsthe ability of the enzyme to accept a properly oriented dUMPfor catalysis and is less sensitive than is K_d to the changesin electrostatics at the phosphate binding site.     

Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase

Arginines R23, R178, R179 and R218 in thymidylate synthase (TS,EC 2.1.1.45) are hydrogen bond donors to the phosphate moietyof the substrate, dUMP. In order to investigate how these argininescontribute to enzyme function, we prepared complete replacementsets of mutants at each of the four sites in Lactobacillus caseiTS. Mutations of R23 increase K_m for dUMP 2–20-fold, increaseK_m for cofactor 8–40-fold and decrease k_cat 9–20-fold,reflecting the direct role of the R23 side chain in bindingand orienting the cofactor in ternary complexes of the enzyme.Mutations of R178 increase K_m for dUMP 40–2000-fold, increaseK_m for cofactor 3–20-fold and do not significantly affectk_cat. These results are consistent with the fact that this residueis an integral part of the dUMP-binding wall and contributesto the orientation and ordering of several other dUMP bindingresidues. Kinetic parameters for all R179 mutations except R179Pwere not significantly different from wild-type values, reflectingthe fact that this external arginine does not directly contactthe cofactor or other ligand-binding residues. R218 is essentialfor the structure of the catalytic site and all mutations ofthis arginine except R218K were inactive.     

The only active mutant of thymidylate synthase D169, a residue far from the site of methyl transfer, demonstrates the exquisite nature of enzyme specificity

Cysteine is the only variant of D169, a cofactor-binding residuein thymidylate synthase, that shows in vivo activity. The 2.4Å crystal structure of Escherichia coli thymidylate synthaseD169C in a complex with dUMP and the antifolate CB3717 showsit to be an asymmetric dimer, with only one active site covalentlybonded to dUMP. At the active site with covalently bound substrate,C169 S     

Recent Developments in PROTAC-Mediated Protein Degradation: From Bench to Clinic

Proteolysis-targeting chimeras (PROTACs), an emerging paradigm-shifting technology, hijacks the ubiquitin-proteasome system for targeted protein degradation. PROTACs induce ternary complexes between an E3 ligase and POI, and this induced proximity leads to polyUb chain formation on substrates and eventual proteasomal-mediated POI degradation. PROTACs have shown great therapeutic potential by degrading many disease-causing proteins, such as the androgen receptor and BRD4. The PROTAC technology has advanced significantly in the last two decades, with the repertoire of PROTAC targets increased tremendously. Herein, we describe recent developments of PROTAC technology, focusing on mechanistic and kinetic studies, pharmacokinetic study, spatiotemporal control of PROTACs, covalent PROTACs, resistance to PROTACs, and new E3 ligands.     

Revisiting the mechanism of the triosephosphate isomerase reaction: the role of the fully conserved glutamic acid 97 residue

An analysis of 503 available triosephosphate isomerase sequences revealed nine fully conserved residues. Of these, four residues—K12, H95, E97 and E165—are capable of proton transfer and are all arrayed around the dihydroxyacetone phosphate substrate in the three‐dimensional structure. Specific roles have been assigned to the residues K12, H95 and E165, but the nature of the involvement of E97 has not been established. Kinetic and structural characterization is reported for the E97Q and E97D mutants of Plasmodium falciparum triosephosphate isomerase (Pf TIM). A 4000‐fold reduction in k_cat is observed for E97Q, whereas the E97D mutant shows a 100‐fold reduction. The control mutant, E165A, which lacks the key catalytic base, shows an approximately 9000‐fold drop in activity. The integrity of the overall fold and stability of the dimeric structure have been demonstrated by biophysical studies. Crystal structures of E97Q and E97D mutants have been determined at 2.0 Å resolution. In the case of the isosteric replacement of glutamic acid by glutamine in the E97Q mutant a large conformational change for the critical K12 side chain is observed, corresponding to a trans‐to‐gauche transition about the Cγ? Cδ (χ³) bond. In the E97D mutant, the K12 side chain maintains the wild‐type orientation, but the hydrogen bond between K12 and D97 is lost. The results are interpreted as a direct role for E97 in the catalytic proton transfer cycle. The proposed mechanism eliminates the need to invoke the formation of the energetically unfavourable imidazolate anion at H95, a key feature of the classical mechanism.     

Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.     

Fluorescence properties of ternary complexes of polymer-bond triphenylphosphine,triphenylarsine, triphenylstibine,and triphenylbismuthine,rare earth metal ions,and thenoyltrifluoroacetone

Rare earth metal ions containing polymer ternary complexes were synthesized and characterized. The functional polymers investigated were polymer-bond triphenylphosphine (PBDP), polymer-bond triphenylarsine (PBDAs), polymer-bond triphenylstibine (PBDSb), and polymer-bond triphenylbismuthine (PBDBi) as polymer ligands. Several substances, such as thenoyltrifluoroacetone (TTA), and 8-hydroxyquinoline (oxin), phenanthroline (phen) were used as low molecular weight ligands. The results show that TTA is the best low molecular weight ligand among them. The fluorescence properties of synthesized complexes were investigated; PBDAs is a better polymer ternary complex that possesses stronger fluorescence intensity coordinated with any low molecular weight ligand. The fluorescence lifetimes of Eu³⁺-containing polymer ternary complexes are between 0.350 and 0.469 MS. The reaction conditions of the formation and stability of rare earth metal ions–polymer–TTA ternary complexes are discussed. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 68: 1605–1611, 1998     

Charge-transfer complexes of 2,3-dichloro-5,6-diyano-p-benzoquinone (D.D.Q.) in solution: a conductimetric study

The formation of charge-transfer complexes has been studied by conductimetric measurements, in the systems diphenylamine-2,3-dichloro-5,6-dicyano-p-benzoquinone (D.D.Q.), 1,10-phenanthroline—D.D.Q., thiourea—D.D.Q. and N : N-dimethylaniline—D.D.Q. in methyl alcohol. The stoichiometry of the charge transfer complexes formed in solution has been determined from the position of the conductivity maximum obtained during the titration of the donor solution with the acceptor solution. The stoichiometry of the diphenylamine—D.D.Q., 1,10-phenanthroline—D.D.Q., thiourea—D.D.Q. and N : N-dimethylaniline—D.D.Q. complexes are 2:1, 1:1, 2:2 and 3:2 respectively. The molar conductivity coefficients for all systems at temperatures of 25, 30, 35 and 40°C have been calculated and are found to increase with the increase in temperature in all the systems studied.     

Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation

We previously demonstrated by X-ray crystallography and electrospraymass spectrometry that D52E mutant hen lysozyme formed a covalentenzyme–substrate adduct on reaction with N-acetylglucosamineoligomer. This observation indicates that D52E lysozyme mayacquire a catalytic pathway via a covalent adduct. To explainthis pathway, the formation and hydrolysis reactions of thecovalent adduct were investigated. Kinetic analysis indicatedthat the hydrolysis step was the rate-limiting step, 60-foldslower than the formation reaction. In the formation reaction,the pH dependence was bell-shaped, which was plausibly explainedby the functions of the two catalytic pK_as of Glu35 and Glu52.On the other hand, the pH dependence in the hydrolysis was sigmoidalwith a transition at pH 4.5, which was identical with the experimentallydetermined pK_a of Glu35 in the covalent adduct, indicating thatGlu35 functions as a general base to hydrolyze the adduct. Toimprove the turnover rate of D52E lysozyme, the mutation ofN46D was designed and introduced to D52E lysozyme. This mutationreduced the activation energy in the hydrolysis reaction ofthe covalent adduct by 1.8 kcal/mol at pH 5.0 and 40°C butdid not affect the formation reaction. Our data may providea useful approach to understanding the precise mechanism ofthe function of natural glycosidases, which catalyze via a covalentadduct.     

Effects of engineered salt bridges on the stability of subtilisin BPN'

Variants designed using PROTEUS have been produced in an attemptto engineer stabilizing salt bridges into subtilisin BPN'. Allthe mutants constructed by site-directed mutagenesis were secretedby Bacillus subtillus, except L75K. Q19E, expressed as a singlevariant and also in a double variant, Q19E/Q271E, appears toform a stabilizing salt bridge based on X-ray crystal structuredetermination and differential scanning calorimeter measurements.Although the double mutant was found to be less thermodynamicallystable than the wild-type, it did exhibit an autolytic stabilityabout two fold greater under hydrophobic conditions. Four variants,A98K, S89E, V26R and L235R, were found to be nearly identicalto wild-type in thermal stability, indicative of stable structureswithout evidence of salt bridge formation. Variants Q271E, V51Kand T164R led to structures that resulted in varying degreesof thermodynamic and autolytic instability. A computer-modelinganalysis of the PROTEUS predictions reveals that the low percentageof salt bridge formation is probably due to an overly simplisticelectrostatic model, which does not account for the geometryof the pairwise interactions.     

A glutamine 67--> histidine mutation in homotetrameric R67 dihydrofolate reductase results in four mutations per single active site pore and causes substantial substrate and cofactor inhibition

R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to increasing clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The crystal structure of R67 DHFR shows a single active site pore that spans the length of the homotetramer. Related sites (due to a 222 symmetry element at the center of the pore) are used to bind ligands, i.e. each half of the pore can accommodate either the substrate, dihydrofolate (DHF), or the cofactor, NADPH, although DHF and NADPH are bound differently. To evaluate the role of glutamine 67 (and its symmetry- related Q167, Q267 and Q367 residues which occur at the center of the active site pore), a Q67H mutation was constructed. Binary binding of dihydrofolate (DHF; monitored by isothermal titration calorimetry) displays two identical sites with a Kd value of 0.04 microM, while binding of NADPH shows two sites possessing negative cooperativity with Kd values of 0.027 and 0.62 microM. A comparison of ligand binding in Q67H versus wild-type (wt) R67 DHFR indicates both ligands bind more tightly (80-6000-fold) and DHF binding in Q67H R67 DHFR no longer displays positive cooperativity as seen in wt R67 DHFR. Ternary complex binding in the Q67H mutant indicates a total of two ligands can bind per pore. Substantial substrate and cofactor inhibition are observed during catalysis, consistent with non-productive binding of either two DHF or two NADPH molecules in Q67H R67 DHFR. Because of the symmetry- related binding sites in the active site pore, the accumulation of potentially positive mutations in R67 DHFR is limited by the balance between tighter binding of ligands (and thus potentially increased catalytic efficiency) and inhibition that arises upon tighter binding of two identical ligands at symmetry-related sites.      

Identification of four amino acid residues essential for catalysis in human cytidine deaminase by site-directed mutagenesis and chemical modifications

By site-directed mutagenesis on human cytidine deaminase (CDA), five mutant proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The three cysteine mutants were completely inactive, whereas E67D and E67Q showed a specific activity about 200- and 200000-fold lower, respectively, than the wild-type CDA. Zinc analysis revealed that only E67D, E67Q and C65A contained 1 mol Zn2+/mol subunit as in the wild- type CDA. Kinetic measurements with the specific carboxylic group reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA suggest that Glu67 is essential for the catalytic process. Furthermore, when both native and denatured CDA was titrated with 5,5'-dithiobis(2-nitrobenzoic acid) six sulfhydryl groups were detected, whereas in the denatured and reduced enzyme nine such groups were found, according to the sequence data. When p-hydroxymercuriphenyl sulfonate was used, nine sulfhydryl groups were detectable and the release of 1 mol of zinc per mole of CDA subunit was revealed by the metal indicator dye 4-(2-pyridylazo)resorcinol. It seems plausible that the limiting step for the maintenance of zinc in the active site is the formation of coordination between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct position and orientation within the active site.      

Contribution of organomodified clay on hybrid microstructures and properties of epoxidized natural rubber‐based nanocomposites

Influence of organic modifying agent of clay on dispersion, distribution, hybrid microstructure formation, and associated performance properties of epoxidized natural rubber‐based composites was evaluated. Binary and ternary composites of carbon black (CB) and two organomodified layered silicates (i.e., nanomer I30E and Cloisite 30B) were prepared and characterized based on small angle X‐ray scattering, transmission electron microscopy, hydrodynamic swelling, tensile measurement, and dynamic mechanical analyses. Greater extent of exfoliation and “nanounit” formation was noted in ternary composites containing nanomer I30E, which was reflected in higher interfacial roughness (d_s = 2.82) and lower radius of gyration (R_g = 205 Å). Morphological observations suggested higher nanomer I30E–CB interactions than that of Cloisite 30B–CB. The interplatelet distance in Cloisite 30B (d = 1.83 nm) stacks was lower than that of nanomer I30E (d = 2.26 nm). These two factors jointly contributed in higher breakdown of nanomer I30E stacks by CB than that of Cloisite 30B stacks. Greater exfoliation and nanounit formation in I30E–CB‐filled nanocomposite was also reflected in increased degree of crosslinking (n = 20 × 10^?5%), tensile modulus/strength, half height width of damping peak (20.3°C), and filler effectiveness (C = 0.33). POLYM. ENG. SCI., 2013. © 2012 Society of Plastics Engineers     

A Comparison of the Binding of the Ligand Trimethoprim to Bacterial and Vertebrate Dihydrofolate Reductases

In this paper, we report on studies of ligand binding to the enzyme dihydrofolate reductase (DHFR). Energy minimizations of four complexes of DHFR with the inhibitor trimethoprim, an antibiotic, and the cofactor NADPH have been carried out in order to investigate the energetics responsible for the 100,000-fold increase in binding affinity of trimethoprim to E. coli DHFR compared with chicken liver DHFR. Several factors suggested to be responsible for the enhanced binding in bacterial DHFR's were investigated in terms of intermolecular and intramolecular energetics. The strain energies of trimethoprim in the four complexes were calculated and found to be about 6 kcal mol⁻¹ in all complexes of the two species. In the binary complex of chicken liver DHFR, where the largest variation was observed, 2 kcal mol⁻¹ higher than in the other complexes, it was found that this increase was compensated for by the slightly more favorable intermolecular interaction of the trimethoxyphenyl moiety with the protein. Comparison of the minimized binary and ternary complexes of E. coli allowed us to investigate the cooperativity in the binding of trimethoprim and NADPH in the bacterial enzyme in terms of the underlying intermolecular forces. This cooperativity was found to be due to a direct trimethoprim - NADPH interaction in the E. coli enzyme rather than enhanced protein-inhibitor interactions induced upon binding of the cofactor. These interactions are not as favorable in the vertebrate enzyme, consistent with the significantly diminished cooperativity observed in this enzyme.     

X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A

The structures of the single-residue mutants H134Q and Y76Aof bovine pancreatic DNase I have been determined and refinedincluding data to 2.3 and 2.4 Å resolution respectively,by X-ray crystallography. H134 is an essential catalytic residue,while Y76 contributes to the binding of DNA by providing a largevan der Waals contact area that stabilizes the wide minor grooveseen in DNase I-DNA complexes. The mutant proteins, which showstrongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A),were expressed in E.coli and both crystallize in space-groupC2 with almost identical unit cells. The crystal packing schemeis different from that found in wild type crystals grown undervery similar conditions, presumably due to the absence of thecarbohydrate moiety. In both mutants the conformation of theprotein is nearly identical to that of the wild type enzymeand changes are confined to surface loops involved in packing.The disruption of the hydrogen bonds between H134, E78 and Y76in both mutants leads to an increased mobility and positionalshifts in the DNA-binding loop, mainly around residue Y76. Thisin turn may further reduce DNA-binding affinity and, thus, contributeto the low activity. In contrast, symmetry contacts involvingresidues 97–108 lead to a stabilization of the flexibleloop compared to wild type DNase I.     

Characterization of cocoa butter substitutes,milk fat and cocoa butter mixtures

The melting properties and polymorphic behavior of binary and ternary blends of cocoa butter substitutes (CBS), cocoa butter (CB), and milk fat (MF) were studied by using pulsed nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and X‐ray diffractometry (XRD). Hydrogenated palm kernel stearin (HPKS) and hydrogenated palm kernel olein (HPKO) were chosen as the two CBS feedstock fats. Both CBS/CB binary blends displayed significant eutectic behaviors. Multiple melting peaks and eutectic effects were observed at 30–50% addition levels of CB to HPKS. The range was broader in HPKO/CB blends. Dilution effect was observed in both CBS/MF blends while slight monotectic effect was also observed in HPKO/MF blends. Ternary phase diagrams and melting curves showed that eutectic effects existed in both ternary blends and the degree of interaction depended on the content of CB and MF. XRD results showed that when pure fat component in each blend exceeded 80%, its polymorphism dominated in the ternary blends. However, when CBS/CB or MF /CB were added at a comparative content (blends D and F), both β and β′ form existed. Practical applications: Phase properties of fat blends may have significant effects on the sensory characteristics and physical properties of the final products, such as hardness, brittleness, and the bloom formation. This study evaluates the melting properties and the polymorphic characteristics of fat blends of constituents with potential use in the manufacture of confectionery products. A comprehensive analysis of binary and ternary fat blends was conducted in order to provide a guide for compound chocolate production formulation.     

Constrained dansyl derivatives reveal bacterial specificity of highly conserved thymidylate synthases

The elucidation of the structural/functional specificities of highly conserved enzymes remains a challenging area of investigation, and enzymes involved in cellular replication are important targets for functional studies and drug discovery. Thymidylate synthase (TS, ThyA) governs the synthesis of thymidylate for use in DNA synthesis. The present study focused on Lactobacillus casei TS (LcTS) and Escherichia coli TS (EcTS), which exhibit 50 % sequence identity and strong folding similarity. We have successfully designed and validated a chemical model in which linear, but not constrained, dansyl derivatives specifically complement the LcTS active site. Conversely, chemically constrained dansyl derivatives showed up to 1000-fold improved affinity for EcTS relative to the inhibitory activity of linear derivatives. This study demonstrates that the accurate design of small ligands can uncover functional features of highly conserved enzymes.     

Interaction of Pestiviral E1 and E2 Sequences in Dimer Formation and Intracellular Retention

Pestiviruses contain three envelope proteins: E^rns, E1, and E2. Expression of HA-tagged E1 or mutants thereof showed that E1 forms homodimers and -trimers. C123 and, to a lesser extent, C171, affected the oligomerization of E1 with a double mutant C123S/C171S preventing oligomerization completely. E1 also establishes disulfide linked heterodimers with E2, which are crucial for the recovery of infectious viruses. Co-expression analyses with the HA-tagged E1 wt/E1 mutants and E2 wt/E2 mutants demonstrated that C123 in E1 and C295 in E2 are the critical sites for E1/E2 heterodimer formation. Introduction of mutations preventing E1/E2 heterodimer formation into the full-length infectious clone of BVDV CP7 prevented the recovery of infectious viruses, proving that C123 in E1 and C295 in E2 play an essential role in the BVDV life cycle, and further support the conclusion that heterodimer formation is the crucial step. Interestingly, we found that the retention signal of E1 is mandatory for intracellular localization of the heterodimer, so that absence of the E1 retention signal directs the heterodimer to the cell surface even though the E2 retention signal is still present. The covalent linkage between E1 and E2 plays an essential role for this process.     

HDPE/ PC/CB复合体系双逾渗行为的研究

以炭黑(CB)为导电填料,填充到2种不相容的高聚物高密度聚乙烯(HDPE)和聚碳酸酯(PC)基体中制备高分子基正温度效应(PTC)材料.研究表明,CB在HIRE中的逾渗阈值约为20%;HDPE/PC/CB三元复合体系形成了双逾渗行为,当HDPE/PC质量比为40/60时,三元复合体系具有较好的PTC及PTC重复性.