Influence of protein flexibility on the redox potential of rubredoxin: Energy minimization studies

A theoretical investigation of the protein contribution to the redox potential of the iron–sulfur protein rubredoxin is presented. Structures of the oxidized and reduced forms of the protein were obtained by energy minimizing the oxidized crystal structure of Clostridium pasteurianum rubredoxin with appropriate charges and parameters. By including 102 crystal waters, structures close to the original crystal structure were obtained (rms difference of 1.16 Å), even with extensive minimization, thus allowing accurate calculations of comparative energies. Our calculations indicate an energy change of about –60 kcal/mol (2.58 eV) in the protein alone upon reduction. This energy change was due to both the change in charge of the redox site and the subsequent relaxation of the protein. An energy minimization procedure for the relaxation gives rms differences between the oxidized and reduced states of about 0.2 Å. The changes were small and occurred in both the backbone and sidechain mainly near the Fe–S center but contributed about – 16 kcal/mol (0.69 eV) to the total protein contribution. Although the neglect of certain effects such as electronic polarization may make the relaxation energies calculated an upper limit, the results indicate that protein relaxation contributes substantially to the redox potential. © 1993 Wiley-Liss, Inc.     

Stereochemical studies on cyclic peptides: Detailed energy minimization studies on hydrogen bonded all-trans cyclic pentapeptide backbones

Conformational studies have been carried out on hydrogenbonded all-trans cyclic pentapeptide backbone. Application of a combination of grid search and energy minimization on this system has resulted in obtaining 23 minimum energy conformations, which are characterized by unique patterns of hydrogen bonding comprising of β- and γ-turns. A study of the minimum energy conformationsvis-a-vis non-planar deviation of the peptide units reveals that non-planarity is an inherent feature in many cases. A study on conformational clustering of minimum energy conformations shows that the minimum energy conformations fall into 6 distinct conformational families. Preliminary comparison with available X-ray structures of cyclic pentapeptide indicates that only some of the minimum energy conformations have formed crystal structures. The set of minimum energy conformations worked out in the present study can form a consolidated database of prototypes for hydrogen bonded backbone and be useful for modelling cyclic pentapeptides both synthetic and bioactive in nature. This is part XV of the series. Part XIV in this series is Ramakrishnanet al 1987.     

Conformational characteristics of the RNA subunit ApA from energy minimization studies

In an attempt to better our understanding of the conformational stabilities in RNAs, an intensive theoraticl study has been carried out on one of its dimeric subunits, ApA, using an improved set of atom-atom interaction energy parameters and an improved version of energy-minimization technique. The C(3′)0endo and the C(2′)-endo sugar ApA units were sperately considered and 38 probable conformations have been analyzed in each case. The total potential energy, comprising nonbonded, electrostatic, and torsional contributions, was minimized by varying all seven relevant dihedral angles simumtaneously. The result reveal that 17 conformations in the case of C(3′)-endo sugar ApA and 7 confomations in the case of C(2′)-endo sugar ApA unit, the lowest energy conformation corresponds to a nonhelical structure and the A-RNA and the Watson-Crick-yype conformations lie at energy levels of about 0.5 and 1.0 Kcal/mo., respectively, above the lowest energy found. For ApA with the lops of different types in the backbone and they all differ in energies by about 3.5 Kcal/mol with refrence to the lowest energy founs. It is noted that the order ofmprefrence of the base stacking is observed in the A-RNA and the Watson-Crick type conformers. The ApA unit with C(2′)-endo sugar is forced to assume phosphodiester conformations with large deviations fom the expected staggered conformations compared to the ApA unit with C(3′)-endo sugar. The result obtained for ApA are discussed with refrence to those previously obtained for the dApdA unit. Te theoretical predictions are compared with the experimental data on the tRNA^Phe crystal, as well as those on fibrous RNAs and RNa subunitlike crystal structures. This study brings out many important aspects of the conformational stability of ApA which have been missed by studies made by others on this system.     

Crystallographic and molecular-orbital studies on the geometry of antifolate drugs.

In the common dihydrofolate reductase inhibitors an amino substituent replaces the pteridine carbonyl oxygen atom of folates, with altered hydrogen-bonding properties and size. Flexibility in the amino groups could facilitate enzyme binding. Studies of cycloguanil hydrochloride by neutron diffraction show both in-plane and out-of-plane deformation of amino groups. Molecular-orbital calculations ab initio on 2,4-diamino-5-methylpyrimidinium cation confirm that the 4-amino group is readily deformable. The 2,4-diaminoquinazoline structure is reported. Atomic co-ordinates, thermal parameters, bond distances and bond angles for cycloguanil and 2,4-diaminoquinazoline have been deposited as Supplementary Publication SUP 50108 (13 pages) at the British Library Lending Division, Boston Spa. Wetherby, West Yorkshire LS23, 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Studies on hydrogen bonds. Part V--Hydrogen bonding in energy minimization studies of peptides

An energy term, representing the N-H...O type of hydrogen bond, which is a function of the hydrogen bond length (R) and angle (theta) has been introduced in an energy minimization program, taking into consideration its interpolation with the non-bonded energy for borderline values of R and theta. The details of the mathematical formulation of the derivatives of the hydrogen bond function as applicable to the energy minimization have been given. The minimization technique has been applied to hydrogen bonded two and three linked peptide units (gamma-turns and beta-turns), and having Gly, Ala and Pro side chains. Some of the conformational highlights of the resulting minimum energy conformations are a) the occurrence of the expected 4----1 hydrogen bond in all of the burn-turn tripeptide sequences and b) the presence of an additional 3----1 hydrogen bond in some of the type I and II tripeptides with the hydrogen bonding scheme in such type I beta-turns occurring in a bifurcated form. These and other conformational features have been discussed in the light of experimental evidence and theoretical predictions of other workers.     

Mechanism of the antimicrobial drug trimethoprim revisited.

We tested the hypothesis that the mechanism of action of the antifolate drug trimethoprim is through accumulation of bacterial dihydrofolate resulting in depletion of tetrahydrofolate coenzymes required for purine and pyrimidine biosynthesis. The folate pool of a strain of Escherichia coli (NCIMB 8879) was prelabeled with the folate biosynthetic precursor [(3)H]-p-aminobenzoic acid before treatment with trimethoprim. Folates in untreated E. coli were present as tetrahydrofolate coenzymes. In trimethoprim-treated cells, however, a rapid transient accumulation of dihydrofolate occurred, followed by complete conversion of all forms of folate to cleaved catabolites (pteridines and para-aminobenzoylglutamate) and the stable nonreduced form of the vitamin, folic acid. Both para-aminobenzoylglutamate and folic acid were present in the cell in the form of polyglutamates. Removal of trimethoprim resulted in the reconversion of the accumulated folic acid to tetrahydrofolate cofactors for subsequent participation in the one-carbon cycle. Whereas irreversible catabolism is probably bactericidal, conversion to folic acid may constitute a bacteriostatic mechanism since, as we show, folic acid can be used by the bacteria and proliferation is resumed once trimethoprim is removed. Thus, the clinical effectiveness of this important drug may depend on the extent to which the processes of either catabolism or folic acid production occur in different bacteria or during different therapeutic regimes.     

基于联合残基力场的蛋白质能量优化

简述了蛋白质结构预测中存在的问题,主要介绍了蛋白质分子力场的一种模型即联合残基力场,然后利用这种模型对一种多肽和葡萄球菌蛋白的部分段进行试验,使它们的能量不同程度的得到了最小化,其中前者的初始能量为-71.50461kcal/mol,最小化后为-73.32767kcal/mol.     

Conformational characteristics of mixed sugar puckered deoxytetranucleoside triphosphate d-GpCpGpC from energy minimization studies

As a continuation of our theoretical studies on nucleic acid subunit systems, in this article we consider the case of the tetranucleoside d-GpCpGpC, the minimally ideal representative unit for analyzing the relative stabilities of different forms of homo- and mixed helical conformation of polynucleotides. The four sugar rings are kept so as to generate B-genus, B+A genus and Z-genus conformations. Twenty five helical conformational states which resulted from judicious mixing of A-, B-, C-, W-, and Z-, states locally are subjected to energy minimization permitting the 19 dihedral angles to vary simultaneously. Conformational states corresponding to regular helical forms and mixed helical forms, when analyzed provide valuable information as to the local conformational flexibility and transitions available to polynucleotides.     

Transfection studies to explore essential folate metabolism and antifolate drug synergy in the human malaria parasite Plasmodium falciparum

Folate metabolism in Plasmodium falciparum is the target of important antimalarial agents. The biosynthetic pathway converts GTP to polyglutamated derivatives of tetrahydrofolate (THF), essential cofactors for DNA synthesis. Tetrahydrofolate can also be acquired by salvage mechanisms. Using a transfection system adapted to studying this pathway, we investigated modulation of dihydropteroate synthase (DHPS) activity on parasite phenotypes. Dihydropteroate synthase incorporates p-aminobenzoate (pABA) into dihydropteroate, the precursor of dihydrofolate. We were unable to obtain viable parasites where the dhps gene had been truncated. However, parasites where the protein was full-length but mutated at two key residues and having < 10% of normal activity were viable in folate-supplemented medium. Metabolic labelling showed that these parasites could still convert pABA to polyglutamated folates, albeit at a very low level, but they could not survive on pABA supplementation alone. This degree of disablement in DHPS also abolished the synergy of the antifolate combination pyrimethamine/sulfadoxine. These data indicate that DHPS activity above a low but critical level is essential regardless of the availability of salvageable folate and formally prove the role of this enzyme in antifolate drug synergy and folate biosynthesis in vivo. However, we found no evidence of a significant role for DHPS in folate salvage. Moreover, when biosynthesis was compromised by the absence of a fully functional DHPS, the parasite was able to compensate by increasing flux through the salvage pathway.     

Conformational analysis of cyclolinopeptide A, a cyclic nonapeptide: nuclear Overhauser effect and energy minimization studies

The conformation of cyclolinopeptide A [cyclo(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring cyclic nonapeptide has been investigated in dimethylsulfoxide solution by 270 MHz 1H-nmr. A complete assignment of all C alpha H and NH resonances has been accomplished using two-dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and JHNC alpha H values permit construction of a molecular model for the cyclic peptide backbone. The crude model derived from nmr has been used as a starting point for energy minimization, which yields a refined structure largely compatible with nmr observations. The major features of the conformation of cyclolinopeptide A are a Type VI beta-turn centered at Pro(1)-Pro(2), with a cis peptide bond between these residues and a gamma-turn (C7 structure) centered at Ile(6). Two intramolecular hydrogen bonds Val(9) CO--Phe(3)NH (4----1) and Leu(5) CO--Ile(7)NH (3----1) are observed in the low-energy conformation. The limited solvent accessibility observed for the Val(9) and Leu(5) NH groups in the nmr studies are rationalized in terms of steric shielding.     

Conformational analysis of phosphatides: Mapping and minimization of the intramolecular energy

Empirical intramolecular energy calculations were carried out on molecular fragments related to phosphatides in order to find the preferred conformations. The energy was mapped as a function of several pairs of torsional angles in progressively larger molecular fragments, with energy minimization being carried out at each map point with respect to other significant variables. The energy mapping results were used as starting points for energy minimization on diheptanoyl L-α-phosphatidic acid-C, which consisted of the named molecule plus a carbon atom attached to one of the phosphate oxygens. It was found that there are 6 pairs of values for 2 of the torsional angles at the 3-way branch point in the glyceryl group which give sterically acceptable conformations; only 4 of these are compatible with lipid bilayer structure in that they can give a parallel arrangement of the acyl chains. The several acceptable conformations of the phosphate and acyl ester groups within each of these conformational classes are enumerated. The results obtained may be used as a guide for further experimental and theoretical work on phosphatide structures.     

Conformational characteristics of dApdA,dApdT, dTpdA,and dTpdT from energy minimization studies

The conformational characteristics of the deoxydinucleoside monophosphates with adenine and thymine bases in all possible sequences, namely, dApdA, dApdT, dTpdA, and dTpdT have been studied using an improved set of energy parameters to calculate the total potential energy and an improved set of energy parameters to calculate the total potential energy and an improved version of the minimization technique to minimize the total energy by allowing all seven dihedral angles of the molecular fragment to vary simultaneously. The results reveal that the most preferred conformation in all these units usually corresponds to one of the four helical conformations, namely, the A-DNA, B-DNA, C-DNA, and Watson-Crick DNA models. These helical conformations differ in energies by about 3 kcal/mol with respect to one another. The conformations which could promote a loop or bend in the backbone are, in general, less stable by about 3.5 kcal/mol with respect to the respective lowest-energy helical conformation. The results indicate that there is a definite influence of bases and their actual sequences on the preferred conformations of the deoxydinucleoside monophosphates. The lowest-energy structure, although corresponding to one of the four helical conformations, differ with the type of the deoxydinucleoside monophosphate. Good or reasonable base stacking is noted in dApdA and dTpdA with both C(3′)-endo and C(2′)-endo sugars and in dApdT and dTpdT with only C(3′)-endo sugar. The inversion of the base sequence in deoxydinucleoside monophosphates alters the order of preference of low-energy conformations as well as the base-stacking property of the unit. The paths linking the starting and final states in the (ω′, ω) plane show interesting features with regard to the energy spread, thus providing insight into the path of conformational movement ofthe molecule under slight perturbation. The stabilities of the A and B forms, including the internal energies of the C(3′)-endo ans C(2′)-endo sugar systems, indicate that for dTpdT the B → A transition is less probable. For dApdA, dApdT, and dTpdA this transition is probable in the same order of preference. We propose that the T-A sequence in the polynucleotide chain might serve as the site accessible for B ? A transitions. The theoretical predictions are in good agreement with the experimental observations.     

Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors

We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and receptor are responsible simultaneously for the preference of inhibition and potency. In this study, fast flexible docking simulations were performed on quinazoline antifolates derivatives as human thymidylate synthase inhibitors. The results indicated that the quinazoline ring of the inhibitors forms hydrophobic contacts with Leu192, Leu221 and Tyr258 and stacking interaction is conserved in complex with the inhibitor and cofactor.     

Conformational characteristics of mixed sugar puckered deoxydinucleoside triphosphate units d-pCpGp and d-pGpCp from energy minimization studies

The deoxydinucleoside triphosphate units d-pCpGp and d-pGpCp were subjected to a rigorous theoretical investigation with a view to describing their distinctive conformational characteristics. For each unit 216 probable three-dimensional forms defined by the backbone-base dihedral angles and sugar pucker modes were considered for conformational energy minimization process and scrutinized with reference to properties, such as base-stacking, hydrogen-bonding, internal flexibility and base sequence-phosphate influence. The P-O bond torsions and the phosphate groups were treated with special attention. The results reveal a number of preferred conformational states other than the known helical forms, such as, A-, B-, C-, Z-, and Watson-Crick conformation. Many interesting one-step (change in only one of the dihedral angles or sugar puckers) conformational transitions which involve just about a kcal/mol of energy came to light. The two base sequences CG and GC were noted to differ strikingly in many of their conformational characteristics.     

Inhibition of histone deacetylase activity is a novel function of the antifolate drug methotrexate

Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor widely used for treating human cancers, and overexpression of histone deacetylase (HDAC) is usually found in tumors. HDAC inhibitors (HDACi) can reactivate tumor suppressor genes and serve as potential anti-cancer drugs. In this study, we found that MTX shared structural similarity with some HDACi and molecular modeling showed that MTX indeed docks into the active site of HDLP, a bacterial homologue of HDAC. Subsequent in vitro assay demonstrated MTX’s inhibition on HDAC activity in human cancer cells. The global acetylation of histone H3 was also induced by MTX. Moreover, MTX inhibited immunoprecipitated HDAC1/2 activity but not their protein levels. This study provides evidence that MTX inhibits HDAC activity.     

Genotoxic evaluation of the antimicrobial drug, trimethoprim, in cultured human lymphocytes

The antimicrobial drug, trimethoprim, was evaluated for genotoxicity in human peripheral blood lymphocyte cultures set-up from two healthy donors. Sister-chromatid exchanges (SCE) and micronuclei (MN) were scored as genetic endpoints. The treatment was done using different trimethoprim concentrations ranging from 1 to 100 microg/ml. From our results, we can conclude that this drug is able to induce both cytotoxic and moderate genotoxic effects, as revealed by the increases seen in SCE and MN frequencies in cultures from the two donors and, at least, at one of the concentrations tested.