Free and bound state structures of 6-O-methyl homoerythromycins and epitope mapping of their interactions with ribosomes

The solution and solid state conformations of several 6-O-methyl homoerythromycins 1–4 were studied using a combination of X-ray crystallography, NMR spectroscopy and molecular modelling calculations. In the solid state 1 was found to exist as the two independent molecules with similar structures termed 3-endo-folded-out. In solution a significant conformational flexibility was noticed especially in the C2 to C5 region. The compounds 1 and 2 unlike 14-membered macrolides adopted the 3-endo-folded-out conformation while 3 and 4 existed in the classical folded-out conformation. TrNOESY and STD experiments showed that 1 and 2 bound to the Escherichia coli ribosome while 3 and 4, lacking the cladinose sugar, did not exhibit binding activities, this being in accordance with biochemical data. The bound conformations were found to be very similar to the free ones, some small differences were observed and discussed. The STD experiments provided evidence on binding epitopes. The structural parts of 1 and 2 in close contact with ribosome were similar, however the degree of saturation transfer was higher for 2. The differences between tr-NOE data and STD enhancements in 1 and 2 arouse as a consequence of structural changes upon binding and a closer proximity of 2 to the ribosome surface. An understanding of the molecular mechanisms involved in the interaction of macrolides with ribosomes can help in developing strategies aiming at design of potential inhibitors.     

Free energy simulations: Applications to the study of liquid water,hydrophobic interactions and solvent effects on conformational stability

Free energy simulations using the Metropolis Monte Carlo method and the coupling parameter approach with umbrella sampling are described for several problems of interest in structural biochemistry; the liquid water, the hydrophobic interaction of alkyl and phenyl groups in water and solvent effects on the conformational stability of the alanine dipeptide and the dimethyl phosphate anion in water. Proximity analysis of results is employed to identify stabilizing factors. Implications of result with respect to the structural chemistry of proteins and nucleic acids is considered.     

Crystal structures of thymidylate synthase mutant R166Q: structural basis for the nearly complete loss of catalytic activity

Thymidylate synthase (TS) catalyzes the folate-dependent methylation of deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). We have investigated the role of invariant arginine 166, one of four arginines that contact the dUMP phosphate, using site-directed mutagenesis, X-ray crystallography, and TS from Escherichia coli. The R166Q mutant was crystallized in the presence of dUMP and a structure determined to 2.9 A resolution, but neither the ligand nor the sulfate from the crystallization buffer was found in the active site. A second structure determined with crystals prepared in the presence of dUMP and the antifolate 10-propargyl-5,8-dideazafolate revealed that the inhibitor was bound in an extended, nonproductive conformation, partially occupying the nucleotide-binding site. A sulfate ion, rather than dUMP, was found in the nucleotide phosphate-binding site. Previous studies have shown that the substitution at three of the four arginines of the dUMP phosphate-binding site is permissive; however; for Arg166, all the mutations lead to a near-inactive mutant. The present structures of TS R166Q reveal that the phosphate-binding site is largely intact, but with a substantially reduced affinity for phosphate, despite the presence of the three remaining arginines. The position of Cys146, which initiates catalysis, is shifted in the mutant and resides in a position that interferes with the binding of the dUMP pyrimidine moiety.     

Free energy calculations on the binding of novel thiolactomycin derivatives to E. coli fatty acid synthase I

Finding novel antibiotics to combat the rise of drug resistance in harmful bacteria is of enormous importance for human health. Computational drug design can be employed to aid synthetic chemists in the search for new potent inhibitors. In recent years, molecular dynamics based free energy calculations have emerged as a useful tool to accurately calculate receptor binding affinities of novel or modified ligands. While being significantly more demanding in computational resources than simpler docking algorithms, they can be employed to obtain reliable estimates of the effect individual functional groups have on protein-ligand complex binding constants. Beta-ketoacyl [acyl carrier protein] synthase I, KAS I, facilitates a critical chain elongation step in the fatty acid synthesis pathway. Since the bacterial type II lipid synthesis system is fundamentally different from the mammalian type I multi-enzyme complex, this enzyme represents a promising target for the design of specific antibiotics. In this work, we study the binding of several recently synthesized derivatives of the natural KAS I inhibitor thiolactomycin in detail based on atomistic modeling. From extensive thermodynamic integration calculations the effect of changing functional groups on the thiolactone scaffold was determined. Four ligand modifications were predicted to show improved binding to the E. coli enzyme, pointing the way towards the design of thiolactomycin derivatives with binding constants in the nanomolar range.     

Aldol additions with mutant lipase: analysis by experiments and theoretical calculations

A Ser105Ala mutant of Candida antarctica lipase B has previously been shown to catalyze aldol additions. Quantum chemical calculations predicted a reaction rate similar to that of natural enzymes, whereas experiments showed a much lower reaction rate. Molecular dynamics simulations, presented here, show that the low reaction rate is a consequence of the low frequencies of near attack complexes in the enzyme. Equilibrium was also considered as a reason for the slow product formation, but could be excluded by performing a sequential reaction to push the reaction towards product formation. In this paper, further experimental results are also presented, highlighting the importance of the entire active site for catalysis.     

Backbone and ILV methyl resonance assignments of E. coli thymidylate synthase bound to cofactor and a nucleotide analogue

Thymidylate synthase (TSase) is a 62 kDa homodimeric enzyme required for de novo synthesis of thymidine monophosphate in most organisms. This makes the enzyme an excellent target for anticancer and microbial antibiotic drugs. In addition, TSase has been shown to exhibit negative cooperativity and half-the-sites reactivity. For these collective reasons, TSase is widely studied, and much is known about its kinetics and structure as it progresses through a multi-step catalytic cycle. Recently, nuclear magnetic resonance spin relaxation has been instrumental in demonstrating the critical role of dynamics in enzyme function in small model systems. These studies raise questions about how dynamics affect function in larger enzymes with more complex reaction coordinates. TSase is an ideal candidate given its size, oligomeric state, cooperativity, and status as a drug target. Here, as a pre-requisite to spin relaxation studies, we present the backbone and ILV methyl resonance assignments of TSase from Escherichia coli bound to a substrate analogue and cofactor.     

Structural and functional analysis of surface domains unique to bacteriophage T4 thymidylate synthase

The bacteriophage T4 genome encodes most of its own enzymes for dNTP synthesis, which form a complex in infected Escherichia coli. The T4 thymidylate synthase (TS) and the T4 deoxycytidylate deaminase (CD) catalyze sequential reactions and are physically linked within this complex [McGaughey, K. M., Wheeler, L. J., Moore, J. T., Maley, G. F. , Maley, F., and Mathews, C. K. (1996) J. Biol. Chem. 271, 23037-23042]. From the crystal structure of T4TS [Finer-Moore, J. S., Maley, G. F., Maley, F., Montfort, W. R., and Stroud, R. M. (1994) Biochemistry 33, 15459-15468], it appears that three regions corresponding to insertions relative to E. coli TS lie on one side of the enzyme surface. We have investigated the residual activity of T4TS in response to complete deletion or substitution mutagenesis of these insertions. Two deletions generated in the small domain (residues 70-103) reduced the TS activity to 0.2% and 0.7% of the wild-type activity, with the latter able to complement growth of a thyA- E. coli strain in the absence of thymidine. By insertion of exogenous sequences variable in length and in sequence into these deletion mutants, enzyme activity increased to 44% that of the wild type. Restoration of the TS activity depended mostly on the hydrophobicity of the inserted residues. The sites of insertions also displayed distinct permissiveness for accommodating the exogenous insertions. Deletions and substitutions near the C-terminus resulted in complete inactivation of the T4TS. Proteolysis experiments revealed that the modified surface loops of the small domain were still accessible and flexible for protein-protein interactions. We have used ELISA to detect a physical association between T4TS and T4CD and compared the binding affinity of WT T4TS for two purified insertion mutants of T4CD. The results obtained showed that the native sequences of the small domain inserts are not required for T4TS/T4CD complex formation.     

Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase

Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants carrying mutant forms of a native acetohydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation. The native gene, A19, was mutated in vitro to create amino acid substitutions at residue 563 or residue 642 of the precursor polypeptide. Transformation with the mutated forms of the A19 gene produced resistance to imidazolinone and sulfonylurea herbicides (563 substitution), or imidazolinones only (642 substitution). The herbicide-resistant phenotype of transformants was also manifested in their in vitro AHAS activity. Seedling explants of both Coker and Acala cotton varieties were transformed with the mutated forms of the A19 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained with the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coker and Acala plants were regenerated from a subset of transformation events. Embryonic cell suspension cultures of both Coker and Acala varieties were biolistically transformed at high frequencies using cloned cotton DNA fragments carrying the mutated forms of the A19 gene. In these transformation experiments the mutated A19 gene served as the selectable marker, and the efficiency of selection was comparable to that obtained with the NPT II gene marker of vector Bin 19. Using this method, transgenic Acala plants resistant to imidazolinone herbicides were obtained. Southern blot analyses indicated the presence of two copies of the mutated A19 transgene in one of the biolistically transformed R₀ plants, and a single copy in one of the R₀ plants transformed with Agrobacterium. As expected. progeny seedlings derived from outcrosses involving the R₀ plant transformed with Agrobacterium segregated in a 1:1 ratio with respect to herbicide resistance. The resistant progeny grew normally after irrigation with 175 g/l of the imidazolinone herbicide imazaquin, which is five times the field application rate. In contrast, untransformed sibling plants were severely stunted.Abbreviations AHAS acetohydroxyacid synthase - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - FW fresh weight - GUS -glucuronidase - IC₅₀ herbicide concentration that produces a 50% reduction in the fresh weight growth of cells - NAA -naphthaleneacetic acid - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962)     

Detection and structure determination of an equilibrium unfolding intermediate of Rd-apocytochrome b562: native fold with non-native hydrophobic interactions

The absence of detectable kinetic and equilibrium folding intermediates by optical probes is commonly taken to indicate that protein folding is a two-state process. However, for some small proteins with apparent two-state behavior, unfolding intermediates have been identified in native-state hydrogen exchange or kinetic unfolding experiments monitored by nuclear magnetic resonance. Rd-apocytochrome b(562), a four-helix bundle, is one such protein. Here, we found another unfolding intermediate for Rd-apocytochrome b(562). It is based on a cooperative transition of (15)N chemical shifts of amide protons as a function of urea concentrations before the global unfolding. We have solved the high-resolution structure of the protein at 2.8 M urea, which is after this cooperative transition but before the global unfolding. All four helices remained intact, but a number of hydrophobic core residues repacked. This intermediate provides a possible structural interpretation for the kinetic unfolding intermediates observed using nuclear magnetic resonance methods for several proteins and has important implications for theoretical studies of protein folding.     

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.     

Interaction of pyridoxal phosphate with thymidylate synthase: Spectral and equilibrium dialysis studies

• 1.1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents.
• 2.2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97.
• 3.3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate.
• 4.4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.

     

Free energy calculations for theophylline binding to an RNA aptamer: Comparison of MM-PBSA and thermodynamic integration methods

We have applied the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method (J. Srinivasan, T. E. Cheatham, P. Cieplak, P. A. Kollman, and D. A. Case, Journal of the American Chemical Society, 1998, Vol. 120, pp. 9401-9409) to study the interaction of an RNA aptamer with theophylline and its analogs. The MM-PBSA free energy analysis provides a reasonable absolute binding free energy for the RNA aptamer-theophylline complex formation. Energetic analysis reveals that the van der Waals interaction and the nonpolar contribution to solvation provide the basis for the favorable absolute free energy of complex. This trend is similar to other protein-ligand interactions studied previously. The MM-PBSA method also ranks the relative binding energies of five theophylline analogs approximately correctly, but not as well as the more conventional thermodynamic integration calculations, which were carried out to convert theophylline into its analogs. The comparison of MM-PBSA with TI suggests that the MM-PBSA method has some difficulties with the first-solvation-shell energetics.     

Human thymidylate synthase with loop 181-197 stabilized in an inactive conformation: ligand interactions, phosphorylation, and inhibition profiles

Thymidylate synthase (TS) is a well-validated cancer target that undergoes conformational switching between active and inactive states. Two mutant human TS (hTS) proteins are predicted from crystal structures to be stabilized in an inactive conformation to differing extents, with M190K populating the inactive conformation to a greater extent than A191K. Studies of intrinsic fluorescence and circular dichroism revealed that the structures of the mutants differ from those of hTS. Inclusion of the substrate dUMP was without effect on M190K but induced structural changes in A191K that are unique, relative to hTS. The effect of strong stabilization in an inactive conformation on protein phosphorylation by casein kinase 2 (CK2) was investigated. M190K was highly phosphorylated by CK2 relative to an active-stabilized mutant, R163K hTS. dUMP had no detectable effect on phosphorylation of M190K; however, dUMP inhibited phosphorylation of hTS and R163K. Studies of temperature dependence of catalysis revealed that the E(act) and temperature optimum are higher for A191K than hTS. The potency of the active-site inhibitor, raltitrexed, was lower for A191K than hTS. The response of A191K to the allosteric inhibitor, propylene diphosphonate (PDPA) was concentration dependent. Mixed inhibition was observed at low concentrations; at higher concentrations, A191K exhibited nonhyperbolic behavior with respect to dUMP and inhibition of catalysis was reversed by substrate saturation. In summary, inactive-stabilized mutants differ from hTS in thermal stability and response to substrates and PDPA. Importantly, phosphorylation of hTS by CK2 is selective for the inactive conformation, providing the first indication of physiological relevance for conformational switching.     

Free energy calculations and binding analysis of two potential anti- influenza drugs with Polymerase basic protein-2 (PB2)

Influenza viruses cause a significant level of morbidity and mortality in the population every year. Their resistance to current anti-influenza drugs increases the difficulty of flu treatment. Thus, development of new anti-influenza drugs is necessary in regards of prevent the tragedy of influenza pandemic. The Polymerase basic protein 2 (PB2) subunit of influenza virus RNA polymerase is one of potential targets for new drugs because the binding of PB2 with the 5' cap of the host pre-mRNAs is the initial step of the virus' protein synthesis. In this study, we compared the binding potency of PB2 cap binding domain with two small molecules, i.e., RO and PPT28, with that of PB2 with cap analog m7GTP. The calculated binding energies showed that RO and PPT28 had higher binding affinity with PB2. Further interaction analysis showed that the important parts for binding were the five- and six-member heterocyclic rings (the 6/5-member rings) of small molecules, as well as the hydrophobic parts of RO and PPT28 which had good interactions with the hydrophobic residues in the binding cavity. Thus, RO and PPT28 are two potential anti-influenza drugs targeted PB2, which may inhibit the growth of influenza virus by competitively binding with the cap structure binding domain of PB2.     

Anti-cooperativity and cooperativity in hydrophobic interactions: Three-body free energy landscapes and comparison with implicit-solvent potential functions for proteins

Potentials of mean force (PMFs) of three-body hydrophobic association are investigated to gain insight into similar processes in protein folding. Free energy landscapes obtained from explicit simulations of three methanes in water are compared with that predicted by popular implicit-solvent effective potentials for the study of proteins. Explicit-water simulations show that for an extended range of three-methane configurations, hydrophobic association at 25 degrees C under atmospheric pressure is mostly anti-cooperative, that is, less favorable than if the interaction free energies were pairwise additive. Effects of free energy nonadditivity on the kinetic path of association and the temperature dependence of additivity are explored by using a three-methane system and simplified chain models. The prevalence of anti-cooperativity under ambient conditions suggests that driving forces other than hydrophobicity also play critical roles in protein thermodynamic cooperativity. We evaluate the effectiveness of several implicit-solvent potentials in mimicking explicit water simulated three-body PMFs. The favorability of the contact free energy minimum is found to be drastically overestimated by solvent accessible surface area (SASA). Both the SASA and a volume-based Gaussian solvent exclusion model fail to predict the desolvation barrier. However, this barrier is qualitatively captured by the molecular surface area model and a recent "hydrophobic force field." None of the implicit-solvent models tested are accurate for the entire range of three-methane configurations and several other thermodynamic signatures considered.     

Crystal structure of recombinant triosephosphate isomerase from Bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three-dimensional structures points to the importance of hydrophobic interactions.

The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2-phosphoglycolate was determined by X-ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR. Twofold averaging and solvent flattening was applied to improve the quality of the map. Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the "closed" conformation in either subunit. The crystallographic R-factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 A for 248 C alpha atoms and have average temperature factors of 18.9 and 15.9 A2, respectively. In both subunits, the active site Lys 10 adopts an unusual phi, psi combination. A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B. stearothermophilus TIM. Although the ratio Arg/(Arg+Lys) is higher in B. stearothermophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stearothermophilus TIM. Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family. Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B. stearothermophilus enzyme. However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B. stearothermophilus TIM compared to all five other known TIM structures.     

Interaction between bound water molecules and local protein structures: A statistical analysis of the hydrogen bond structures around bound water molecules

Water molecules play an important role in protein folding and protein interactions through their structural association with proteins. Examples of such structural association can be found in protein crystal structures, and can often explain protein functionality in the context of structure. We herein report the systematic analysis of the local structures of proteins interacting with water molecules, and the characterization of their geometric features. We first examined the interaction of water molecules with a large local interaction environment by comparing the preference of water molecules in three regions, namely, the protein–protein interaction (PPI) interfaces, the crystal contact (CC) interfaces, and the non‐interfacial regions. High preference of water molecules to the PPI and CC interfaces was found. In addition, the bound water on the PPI interface was more favorably associated with the complex interaction structure, implying that such water‐mediated structures may participate in the shaping of the PPI interface. The pairwise water‐mediated interaction was then investigated, and the water‐mediated residue–residue interaction potential was derived. Subsequently, the types of polar atoms surrounding the water molecules were analyzed, and the preference of the hydrogen bond acceptor was observed. Furthermore, the geometries of the structures interacting with water were analyzed, and it was found that the major structure on the protein surface exhibited planar geometry rather than tetrahedral geometry. Several previously undiscovered characteristics of water–protein interactions were unfolded in this study, and are expected to lead to a better understanding of protein structure and function. Proteins 2016; 84:43–51. © 2015 Wiley Periodicals, Inc.