Functionally orthogonal ligand-receptor pairs for the selective regulation of gene expression generated by manipulation of charged residues at the ligand-receptor interface of ER alpha and ER beta

The reengineering of protein-small molecule interfaces represents a powerful tool of chemical biology. For many applications it is necessary to engineer receptors so that they do not interact with their endogenous ligands but are highly responsive to designed ligand analogues, which in turn do not interact with endogenous proteins. The chemical design strategy used to reengineer protein-small molecule interfaces is particularly challenging for interfaces involving relatively plastic receptor binding sites and therefore presents a unique challenge in molecular design. In this study we explore the scope and limitations of a new strategy for manipulating polar/charged residues across the ligand receptor interface of estradiol (E2) and the estrogen receptor (ER). Carboxylate-functionalized E2 analogues can activate ER alpha(Glu353-->Ala) and ER beta(Glu305-->Ala) with very large selectivites, demonstrating that this design strategy is extendable to other members of the steroid hormone receptor family. Neutral E2 analogues were found to complement ER alpha(E353A) with similar potencies but with generally lower selectivities. This suggests that the high selectivity observed with ligand-receptor pairs generated by exchanging charged residues across ligand-receptor interfaces is only due in part to their complementary shapes and that appropriate introduction of charged functionality on the ligand can provide substantial enhancement of selectivity by decreasing the engineered ligands affinity for the endogenous receptor. Attempts to modify the cationic residues by complementing Arg394-->Ala or Arg394-->Glu were not successful.     

Lone-pair orbital interactions in polythiaadamantanes

The electronic structures of a series of polythiaadamantanes from thiaadamantane through 2,4,6,8,9,10-hexathiaadamantane (HTA) have been analyzed using density functional theory calculations in conjunction with Hückel and natural bond orbital analysis. The effects of multiple sulfur p-type lone-pair orbital interactions on ionization potentials, hole mobilities, and electronic coupling have been determined. An overall increase in the average energy of the lone-pair orbitals as the number of sulfur atoms increases is predicted, with the exact positioning of the HOMO depending on specific lone-pair interactions. Separation of through-bond (TB) and through-space (TS) interactions between intramolecular sulfur atoms has been performed using localized molecular orbitals and model systems based on interacting hydrogen sulfide molecules. TB interations were found to reduce orbital splitting, while TS interactions were found to increase orbital splitting. TS interactions were more or less constant from one polythiaadamantane to the next, and the contributions of TB effects to individual orbital energies vary depending on the relative orientation of sulfur atoms as determined by the sigma molecular framework. Electronic coupling between intermolecular sulfur lone-pair orbitals was determined by investigating unique dimer pairs observed in the crystal structure of HTA. Electronic coupling is not as strong as expected given the short intermolecular S-S distances observed in the crystal structure. In general, B3LYP/6-31G(d) and B3LYP/6-311+G(d,p) give very similar orbital energies and splittings.     

Why do the heavy-atom analogues of acetylene E2H2 (E = Si-Pb) exhibit unusual structures?

DFT calculations at BP86/QZ4P have been carried out for different structures of E(2)H(2) (E = C, Si, Ge, Sn, Pb) with the goal to explain the unusual equilibrium geometries of the heavier group 14 homologues where E = Si-Pb. The global energy minima of the latter molecules have a nonplanar doubly bridged structure A followed by the singly bridged planar form B, the vinylidene-type structure C, and the trans-bent isomer D1. The energetically high-lying trans-bent structure D2 possessing an electron sextet at E and the linear form HEEH, which are not minima on the PES, have also been studied. The unusual structures of E(2)H(2) (E = Si-Pb) are explained with the interactions between the EH moieties in the (X(2)Pi) electronic ground state which differ from C(2)H(2), which is bound through interactions between CH in the a(4)Sigma(-) excited state. Bonding between two (X(2)Pi) fragments of the heavier EH hydrides is favored over the bonding in the a(4)Sigma(-) excited state because the X(2)Pi --> a(4)Sigma(-) excitation energy of EH (E = Si-Pb) is significantly higher than for CH. The doubly bridged structure A of E(2)H(2) has three bonding orbital contributions: one sigma bond and two E-H donor-acceptor bonds. The singly bridged isomer B also has three bonding orbital contributions: one pi bond, one E-H donor-acceptor bond, and one lone-pair donor-acceptor bond. The trans-bent form D1 has one pi bond and two lone-pair donor-acceptor bonds, while D2 has only one sigma bond. The strength of the stabilizing orbital contributions has been estimated with an energy decomposition analysis, which also gives the bonding contributions of the quasi-classical electrostatic interactions.     

Comparison of a 3D-model of the classical α-scorpion toxin V from Leiurus quinquestriatus quinquestriatus with other scorpion toxins

In the present paper, a study of classical and insect alpha-scorpion toxins is described. A homology model of the classical alpha-toxin LqqV from Leiurus quinquestriatus quinquestriatus was developed. The model was compared to stable and energetically favourable conformations of AaHII from Androctonus australis Hector and LqhalphaIT from Leiurus quinquestriatus hebraeus, which are the most active alpha-toxins in mammals and insects. The conformations were retrieved from molecular dynamics simulations of known structures. The model of LqqV shows a C-terminal conformation similar to LqhalphaIT. This is mainly caused by electrostatic interactions between Lys10 /Lys60 and Glu59, which are comparable to the cation-pi interactions of Tyr10 and Arg64 in LqhalphaIT. During the simulations the structures of AaHII and LqqV were stabilised through electrostatic interactions between Glu32 and Lys50 and especially the loop adjacent to the alpha-helix is affected, which is in contrast to LqhalphaIT. When the molecular electrostatic potentials of the toxins were studied, a possibly important difference between the classical alpha-toxins and the insect alpha-toxin LqhalphaIT was found in the area around Lys30 and Arg56 of AaHII, where a positive potential is missing in LqhalphaIT. A large negative potential caused by Asp3, Glu15 and Asp19 in LqhalphaIT is also unique for this toxin. It is proposed that Arg18, which is important for activity of LqhalphaIT, restricts the negative potential in this area and is not essential for toxins where negatively charged residues in comparable positions are not present.     

All electron quantum chemical calculation of the entire enzyme system confirms a collective catalytic device in the chorismate mutase reaction

To elucidate the catalytic power of enzymes, we analyzed the reaction profile of Claisen rearrangement of Bacillus subtilis chorismate mutase (BsCM) by all electron quantum chemical calculations using the fragment molecular orbital (FMO) method. To the best of our knowledge, this is the first report of ab initio-based quantum chemical calculations of the entire enzyme system, where we provide a detailed analysis of the catalytic factors that accomplish transition-state stabilization (TSS). FMO calculations deliver an ab initio-level estimate of the intermolecular interaction between the substrate and the amino acid residues of the enzyme. To clarify the catalytic role of Arg90, we calculated the reaction profile of the wild-type BsCM as well as Lys90 and Cit90 mutant BsCMs. Structural refinement and the reaction path determination were performed at the ab initio QM/MM level, and FMO calculations were applied to the QM/MM refined structures. Comparison between three types of reactions established two collective catalytic factors in the BsCM reaction: (1) the hydrogen bonds connecting the Glu78-Arg90-substrate cooperatively control the stability of TS relative to the ES complex and (2) the positive charge on Arg90 polarizes the substrate in the TS region to gain more electrostatic stabilization.     

Electronic structure, molecular electrostatic potential and hydrogen bonding in DMSO-X complexes (X = ethanol, methanol and water)

In the present work, we have studied the electronic structure, molecular electrostatic potential (MEP) and hydrogen bonding in DMSO-ethanol, DMSO-methanol and DMSO-water complexes by employing the MP2 method. Different conformers were simulated on the basis of possible binding sites guided by molecular electrostatic potential topology. The stronger hydrogen bonded interaction lowers the energy of the conformer. Molecular electron density topology and natural bond orbital analysis were used to explain the strength of interactions. Experimental vibrations are also compared with the calculated normal vibrations. Blue shift is predicted for SC vibration in experimental and theoretical spectra as well. Molecular electrostatic potential and topology are used to understand the interaction strength of the conformer.     

DNA and estrogen receptor interaction revealed by fragment molecular orbital calculations

Molecular orbital calculations of the complex between DNA-ERE (estrogen response element) and ER (estrogen receptor)-DBD (DNA-binding domain) were performed using the fragment molecular orbital (FMO) method, which enables large-scale MO (molecular orbital) calculations by reducing the computational cost and by significantly increasing efficiency for parallel computation. Such a large system, which contains 3354 atoms, is impractical via conventional MO methods due to the immense computational cost. Details of the interaction between DNA-ERE and ER-DBD were revealed in this study as follows by using the FMO calculations to analyze the interfragment interaction energies (IFIEs) and the electrostatic potentials (ESPs). An area with a high positive ESP is identified on the DNA-binding side of ER-DBD and is the main driving force behind access to the DNA. The position of the ER-DBD monomer can be fixed on a phosphate group of DNA-ERE by the strong electrostatic interactions, whereas the rotation cannot be fixed. In contrast, both the position and rotation of the ER-DBD dimer can be fixed and can therefore form the stable (ER-DBD)2...DNA-ERE complex. Dimerization of the ER-DBD monomers, each of which have a charge of +5 , is mainly due to large attractive interaction energies of the second Zn fragments. The base pairs in the consensus sequence of DNA-ERE interact only with the recognition helix located in the major groove due to the large shielding effect of the phosphate groups of DNA. The recognition helix has weaker interactions with the base pairs than the electrostatic interactions with the phosphate groups. Thus, the DNA-binding machinery of the ER-DBD dimer, which can secure the recognition helix in the major groove of DNA, is crucial for interactions between the recognition helix and base pairs.     

Intra- and intermolecular interactions between cyclic-AMP receptor protein and DNA: ab initio fragment molecular orbital study

The ab initio fragment molecular orbital (FMO) calculations were performed for the cAMP receptor protein (CRP) complexed with a cAMP and DNA duplex to elucidate their sequence-specific binding and the stability of the DNA duplex, as determined by analysis of their inter- and intramolecular interactions. Calculations were performed with the AMBER94 force field and at the HF and MP2 levels with several basis sets. The interfragment interaction energies (IFIEs) were analyzed for interactions of CRP-cAMP with each base pair, DNA duplex with each amino acid residue, and each base pair with each residue. In addition, base-base interactions were analyzed including hydrogen bonding and stacking of DNA. In the interaction between DNA and CRP-cAMP, there was a significant charge transfer (CT) from the DNA to CRP, and this CT interaction played an important role as well as the electrostatic interactions. It is necessary to apply a quantum mechanical approach beyond the "classical" force-field approach to describe the sequence specificity. In the DNA intramolecular interaction, the dispersion interactions dominated the stabilization of the base-pair stacking interactions. Strong, attractive 1,2-stacking interactions and weak, repulsive 1,3-stacking interactions were observed. Comparison of the intramolecular interactions of free and complexed DNA revealed that the base-pairing interactions were stronger, and the stacking interactions were weaker, in the complexed structure. Therefore, the DNA duplex stability appears to change due to both the electrostatic and the CT interactions that take place under conditions of DNA-CRP binding.     

Identification of the estrogen receptor Cd-binding sites by chemical modification

The widely reported interactions of the estrogen receptor (ER) with endocrine disrupting chemicals (EDCs) present in the environment gave raise to public concern and led to a number of screening and testing initiatives on the international level. Recent studies indicated that certain heavy metals, including cadmium, can mimic the effects of the endogenous estrogen receptor agonist 17beta-estradiol, and lead to estrogen receptor activation. Previous studies of the chimeric proteins, which incorporate the ligand-binding domain of the human ER, identified Cys 381, Cys 447, Glu 523, His 524 and Asp 538 as possible sites of interactions with cadmium. In the present study we utilized the rainbow trout ER ligand-binding domain fused to glutathione-S-transferase, and used Cd-shielding against various types of chemical modification of the fusion protein to study non-covalent interactions between the ER and Cd. The distribution of exposed and shielded residues allowed to identify amino acid residues involved in the interaction. Our data indicated preferential protection of Cys groups by cadmium, suggesting their involvement in the interaction. This supports data found in the literature on the strong binding affinity of the thiol group towards metals. However, not all Cys in the fusion protein sequence were protected against chemical modification, illustrating the importance of their chemical environment. In general, the location of rtER-LBD Cys residues implicated in Cd interactions did not confirm assignments made by alanine-scanning mutagenesis for the hER, probably due to differences in experimental setup and fusion proteins used. The involvement of other functional groups such as carboxylic acids in the Cd interactions, though not confirmed, can not be completely ruled out due to the general limitations of the chemical modification approach discussed in detail. Suggestions for an improved experimental setup were made.     

Comparative analysis of binding energy of chymostatin with human cathepsin A and its homologous proteins by molecular orbital calculation

Cathepsin A is a mammalian lysosomal enzyme that catalyzes the hydrolysis of the carboxy-terminal amino acids of polypeptides and also regulates beta-galactosidase and neuraminidase-1 activities through the formation of a multienzymic complex in lysosomes. Human cathepsin A (hCathA), yeast carboxypeptidase (CPY), and wheat carboxypeptidase II (CPW) belong to the alpha/beta-hydrolase fold family. They have structurally similar active-site clefts, but there are small differences in the amino acid residues comprising their active sites that might determine the substrate specificity and sensitivity to microbial inhibitors including chymostatin. To examine the selectivity and binding mechanism of chymostatin as to hCathA, CPY, and CPW at the atomic level, we analyzed the interaction energy between chymostatin and each protein quantitatively by semiempirical molecular orbital calculation AM1 with the continuum solvent model. We predicted the electrostatic repulsion between the P3 cyclic arginine residue of the inhibitor and the Arg344 in the S3 active subsite of hCathA. Genetic conversion of Arg344 of the wild-type hCathA to Ile also caused an increase in its sensitivity to chymostatin, which was correlated with the decrease in the interaction energy calculated with the molecular orbital method. The present results suggest that such molecular calculation should be useful for evaluating the interactions between ligands, including inhibitors and homologous enzymes, in their docking models.     

On the use of isovalued surfaces to determine molecule shape and reaction pathways

Summary Novel insights into local molecule structure and reactivity can be gained from viewing isovalued surfaces of the molecular electron density, electrostatic potential and molecular orbitals rendered as colored, 3-D objects. For example, drawing positive and negative electrostatic isopotential surfaces partitions the molecule into regions subject to nucleophilic or electrophilic attack. Similarly, coloring isodensity surfaces to indicate the magnitude of the gradient of the electron density maps the molecule surface into regions of high and low electronegativity.A basic understanding of reaction mechanisms can also come from viewing and manipulating isovalued surfaces. A theory of molecular interactions, based upon second-order perturbation theory, provides for the decomposition of the intermolecular interaction energy into steric, electrostatic and orbital interactions. Color figures illustrate the docking of reactant molecular densities, electrostatic potentials and orbitals on low-energy pathways. The figures are used to visualize the steric, electrostatic and orbital contributions to molecular interaction energy. The visualization not only identifies low-energy reaction pathways, but it frequently reveals local interactions which determine the magnitude of the total interaction energy. Similar insight is not easily obtained by simple evaluation of the total interaction energy. Approximate transition states, built from structures along low-energy approach pathways, are excellent starting points for transition state searches.CAChe^TM Technical Report No. 1, Tektronix Inc., Beaverton, OR.     

An analysis of the through-bond interaction using the localized molecular orbitals with ab initio calculations—II : Lone-pair orbital energies in bicyclo compounds: 7-azabicyclo[2.2.1]heptane and 2-azabicyclo[2.2.2]octane,and their n-methyl derivatives

Ab intio SCF MO calculations using STO-3G basis set were performed on 7-azabicyclo[2.2.1]heptane, N-methyl-7-azabicyclo[2.2.1]heptane, 2-azabicyclo[2.2.2)octane, N-methyl-2-azabicyclo[2.2.2)octane, and their model molecules. The orbital energies obtained by these calculations were compared with the experimental ionization potentials The canonical MOs obtained for the model molecules were then transformed into the localized Mos. With the use of the localized MOs thus obtained, the lone-pair orbital energies were pursued in the light of the through-space and/or the through-bond interactions between thw specified localized MOs. As a result of this analysis, it was found that the effects of the inner shell orbitals, 1s electrons of the N atom, and of the neighbouring N-C bonds of the skeleton (through-bond interaction) play a dominant role in the interaction with the lone-pair orbitals. It was also found that the effect of the N-Me group on the lone-pair orbital energy is considerably important.     

Amino acid adsorption on mesoporous materials: influence of types of amino acids, modification of mesoporous materials, and solution conditions

In order to disclose the dominant interfacial interaction between amino acids and ordered mesoporous materials, the adsorption behaviors of five amino acids on four mesoporous materials were investigated in aqueous solutions with adjustable amino acid concentration, ion strength, and pH. The selected amino acids were acidic amino acid glutamic acid (Glu), basic amino acid arginine (Arg), and neutral amino acids phenylalanine (Phe), leucine (Leu), and alanine (Ala), and the selected mesoporous materials were SBA-15, Al-SBA-15, CH3(10%)-SBA-15, and CH3(20%)-SBA-15. The adsorption capacities of Glu and Arg were strongly dependent on pH and surface charge of the mesoporous adsorbent. The adsorption of Phe showed pH insensitivity but depended on the surface organic functionalization of mesoporous adsorbent. On the basis of the theoretical analysis about the interaction between amino acid and adsorbent, such a remarkable difference was attributed to the different nature of the interaction between amino acid and adsorbent. Arg could be readily adsorbed on the surface of SBA-15, especially Al-SBA-15, under appropriate pH in which the electrostatic interaction was predominant. The driving force of Phe adsorption on mesoporous adsorbent mainly came from the hydrophobic interaction. Therefore, the adsorption capability of Arg decreased with increasing ion strength of solution, while the adsorption capability of Phe increased with the increasing degree of CH3 functionalization on SBA-15. For neutral amino acid Phe, Ala, and Leu, the adsorption capability increased with the increase of the length of their side chains, which was another evidence of hydrophobic effect. Thus, all the adsorption of amino acids on mesoporous silica materials can be decided by the combined influence of two fundamental interactions: electrostatic attraction and hydrophobic effect.     

The chemical nature of the (plus sign in circle)C-H...X- (X=Cl or Br) interaction in imidazolium halide ionic liquids

The C-H...X (X=Cl or Br) interaction is traditionally characterized as a relatively weak interaction. However, this interaction becomes very strong in the imidazolium-based halide ionic liquids [J. Phys. Chem. 123, 174501 (2005)]. This strong interaction had been attributed to the electrostatic interaction between the imidazolium cation and the halide anion. In this paper, the chemical nature of the (plus sign in circle)C-H...Cl(-) and ( plus sign in circle)C-H...Br(-) interactions is investigated by atoms in molecules (AIM) and natural bond orbital (NBO) analyses. The AIM calculations indicate that in the EmimX complexes, the (plus sign in circle)C-H...Cl(-) and (plus sign in circle)C-H...Br(-) interactions have some covalent character, especially the (plus sign in circle)C-H...Cl(-) interaction. Mulliken, ChelpG charge, and natural bond orbital population analyses for these two kinds of interactions indicate that the charge transfer is important in the interaction of the cation with the anion. In addition, the NBO analysis demonstrated that the stabilization energy is due to an n-->sigma(C-H) (*) orbital interaction. However, in the Emim2X and Emim3X complexes, the calculated results suggested a dominant electrostatic character for the (plus sign in circle)C-H...Cl(-) and (plus sign in circle)C-H...Br(-) interactions.     

Empirical formulation and parameterization of cation-π interactions for protein modeling

Cation-π interaction is comparable and as important as other main molecular interaction types, such as hydrogen bond, electrostatic interaction, van der Waals interaction, and hydrophobic interaction. Cation-π interactions frequently occur in protein structures, because six (Phe, Tyr, Trp, Arg, Lys, and His) of 20 natural amino acids and all metallic cations could be involved in cation-π interaction. Cation-π interactions arise from complex physicochemical nature and possess unique interaction behaviors, which cannot be modeled and evaluated by existing empirical equations and force field parameters that are widely used in the molecular dynamics. In this study, the authors present an empirical approach for cation-π interaction energy calculations in protein interactions. The accurate cation-π interaction energies of aromatic amino acids (Phe, Tyr, and Try) with protonated amino acids (Arg and Lys) and metallic cations (Li(+), Na(+), K(+), and Ca(2+)) are calculated using B3LYP/6-311+G(d,p) method as the benchmark for the empirical formulization and parameterization. Then, the empirical equations are built and the parameters are optimized based on the benchmark calculations. The cation-π interactions are distance and orientation dependent. Correspondingly, the empirical equations of cation-π interactions are functions of two variables, the distance r and the orientation angle θ. Two types of empirical equations of cation-π interactions are proposed. One is a modified distance and orientation dependent Lennard-Jones equation. The second is a polynomial function of two variables r and θ. The amino acid-based empirical equations and parameters provide simple and useful tools for evaluations of cation-π interaction energies in protein interactions.     

Physical nature of interactions within the active site of cytosine-5-methyltransferase

The physical nature of interactions within the active site of cytosine-5-methyltransferase (CMT) was studied using a variation-perturbation energy decomposition scheme defining a sequence of approximate intermolecular interaction energy models. These models have been used to analyze the catalytic activity of residues constituting cytosine-5-methyltransferase active site as well their role in the binding group of de novo designed inhibitors. Our results indicate that Glu119, Arg163, and Arg165 appear to play the dominant role in stabilizing the protonated transition state structure and their influence can be qualitatively approximated by electrostatic interactions alone. The stabilization of neutral structures of the alternative reaction pathway is small, which might suggest the protonated pathway as preferred by the enzyme. Exchange and delocalization terms are negligible in most cases, or they cancel each other to some extent. Interactions of inhibitors with the CMT active site are dominated by electrostatic multipole contributions in analogy with previously studied transition state analogue inhibitors of leucyl aminopeptidase.     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.