Biomedical subjects

Maria J Ramos

Publications and source records attributed to Maria J Ramos.

At least 19 recordsLinked to original sources

PubMed2007

Computational alanine scanning mutagenesis--an improved methodological approach.

Alanine scanning mutagenesis of protein-protein interfacial residues can be applied to a wide variety of protein complexes to understand the structural and energetic characteristics of the hot-spots. Binding free energies have been estimated with reasonable accuracy with empirical methods, such as Molecular Mechanics/Poisson-Boltzmann surface area (MM-PBSA), and with more rigorous computational approaches like Free Energy Perturbation (FEP) and Thermodynamic Integration (TI). The main objective of this work is the development of an improved methodological approach, with less computational cost, that predicts accurately differences in binding free energies between the wild-type and alanine mutated complexes (DeltaDeltaG(binding)). The method was applied to three complexes, and a mean unsigned error of 0.80 kcal/mol was obtained in a set of 46 mutations. The computational method presented here achieved an overall success rate of 80% and an 82% success rate in residues for which alanine mutation causes an increase in the binding free energy > 2.0 kcal/mol (warm- and hot-spots). This fully atomistic computational methodological approach consists in a computational Molecular Dynamics simulation protocol performed in a continuum medium using the Generalized Born model. A set of three different internal dielectric constants, to mimic the different degree of relaxation of the interface when different types of amino acids are mutated for alanine, have to be used for the proteins, depending on the type of amino acid that is mutated. This method permits a systematic scanning mutagenesis of protein-protein interfaces and it is capable of anticipating the experimental results of mutagenesis, thus guiding new experimental investigations.

Alanine↗

PubMed2006

Insights on resistance to reverse transcriptase: the different patterns of interaction of the nucleoside reverse transcriptase inhibitors in the deoxyribonucleotide triphosphate binding site relative to the normal substrate.

It is presently known that the long-term failure in the treatment of AIDS with the currently available nucleotide reverse transcriptase inhibitors (NRTIs) is related to the development of resistance by reverse transcriptase (RT) at the binding or incorporation level or both, or subsequent to the nucleotide incorporation (excision). To achieve greater insight on the differential interactions of two NRTIs that are mainly discriminated by different mechanisms, 2',3'-didehydro-2',3'-dideoxythymidine-5'-triphosphate (d4TTP, that is, phosphorylated stavudine) and 2',3'-dideoxycytidine-5'-triphosphate (ddCTP, that is, phosphorylated zalcitabine), with the primer/template (p/t) and with the N binding site of reverse transcriptase (RT) in relation to the normal substrate (dNTP), we have conducted a series of molecular dynamics (MD) simulations. We propose that the different resistance profiles arise from the different conformations adopted by the inhibitors at the N site. d4TTP adopts an ideal conformation for catalysis because it forms an ion-dipole intramolecular interaction with the beta-phosphate oxygen of the triphosphate, as does the normal substrate. In ddCTP, the lack of this essential interaction results in a different, noncatalytic conformation.

Binding Sites↗

PubMed2006

QM/QM study of the coverage effects on the adsorption of amino-cyclopentene at the Si(100) surface.

In this work, we have tested 30 different adsorption situations in several coverage scenarios for the 1-amino-3-cyclopentene (ACP) molecule on the Si(100) surface. We have used a five-spot testing zone inserted in the high-level part of a quantum-mechanical/quantum-mechanical study performed in a big cluster. By defining several different scenarios, each one with a typical adsorption energy, we were able to understand in detail the process of surface functionalization. We are able to justify why the functionalization of this silicon surface achieves only a coverage of approximately 0.5 ML (half monolayer) and why the completely covered surface should be thermodynamically impossible to obtain.

Journal Article↗

PubMed2006

Enzyme ribonucleotide reductase: unraveling an enigmatic paradigm of enzyme inhibition by furanone derivatives.

Several 2'-substituted-2'-deoxyribonucleotides are potent inactivators of the enzyme ribonucleotide reductase (RNR), by destroying the essential tyrosyl radical located in subunit R2 or/and covalently alkylating the subunit R1. In the absence of external reductants, the inactivation is achieved by alkylation of subunit R1 by a methylene-3(2H)-furanone. The furanone is generated in solution through degradation of a keto-deoxyribonucleotide intermediate, produced during the inhibitory mechanism of a wide group of 2'-substituted inhibitors, and is easily detected experimentally by UV spectroscopy. Interestingly, the same keto-deoxyribonucleotide is also a proposed intermediate of the normal substrate pathway, but by some unknown reason, it does not dissociate from the active site and does not inactivate the enzyme. Therefore, if the currently accepted mechanism for substrate reduction is correct, there must be some specific reason that makes such a reactive intermediate behave differently, not dissociating from the active site during substrate reduction. In this article, we propose to validate the current substrate mechanism by showing that the keto-deoxyribonucleotide dissociates from the active site only in the case of the inhibitors, and therefore, it corresponds to a viable intermediate in the substrate mechanism. Furthermore, we answer unexplained experimental observations that concern the predomination of the normal reduction mechanism over the abnormal ketone formation in the FdNDP and the release of F(-), either in the normal or in the abnormal turnover. For that purpose, we have investigated the interaction between the enzyme and this keto-deoxyribonucleotide generated from the normal substrate and from two widely studied representative inhibitors. A model containing 140 atoms was used to represent the desired structures. The results allowed us to conclude that the solvation free energy of the 2'-substituents, its influence inside the active site, and the charge transfer mechanism from a protein side chain to solution are the thermodynamic driving forces for the intermediate dissociation and subsequent RNR inhibition. Such charge transfer cannot be accomplished by the natural substrate, preventing its dissociation. These results elucidate a paradox which has been unexplained for more than 20 years and further validates both the proposed substrate and inhibition chemical mechanisms.

Binding Sites↗

PubMed2006

Molecular dynamics simulations of the enzyme Cu, Zn superoxide dismutase.

The enzyme Cu, Zn superoxide dismutase (Cu,Zn-SOD) is a ubiquitous oxireductase, which is responsible for the cellular defense against oxidative stress caused by the high toxicity of the superoxide radical, and has been also linked to some cases of familiar amyotrophic lateral sclerosis. In the present study a set of molecular mechanics parameters for the active site of Cu,Zn-SOD has been derived. Afterward, an extensive molecular dynamics simulation has been carried out in an aqueous environment. The obtained results shed a further light on the structural flexibility of the backbone, where the active site is nested, and the solvation shell occupancy. The relatively small backbone deviation, shown by a root-mean-square deviation below 1.0 A, confirms the accuracy of the parameters. The solvent shell analysis has shown that the first solvation shell is located at about 5 A from the copper ion, generating an empty cavity with enough space to accommodate the superoxide radical. The low residence time means that a high permutation rate of water molecules in both solvation shells is consistent with the efficiency of this catalytic mechanism. Hybrid studies using ONIOM methodologies can now be done to evaluate the mechanistic implications of the explicit inclusion of the whole system.

Computer Simulation↗

PubMed2006

Comparative evolutionary genomics of the HADH2 gene encoding Abeta-binding alcohol dehydrogenase/17beta-hydroxysteroid dehydrogenase type 10 (ABAD/HSD10).

BACKGROUND: The Abeta-binding alcohol dehydrogenase/17beta-hydroxysteroid dehydrogenase type 10 (ABAD/HSD10) is an enzyme involved in pivotal metabolic processes and in the mitochondrial dysfunction seen in the Alzheimer's disease. Here we use comparative genomic analyses to study the evolution of the HADH2 gene encoding ABAD/HSD10 across several eukaryotic species. RESULTS: Both vertebrate and nematode HADH2 genes showed a six-exon/five-intron organization while those of the insects had a reduced and varied number of exons (two to three). Eutherian mammal HADH2 genes revealed some highly conserved noncoding regions, which may indicate the presence of functional elements, namely in the upstream region about 1 kb of the transcription start site and in the first part of intron 1. These regions were also conserved between Tetraodon and Fugu fishes. We identified a conserved alternative splicing event between human and dog, which have a nine amino acid deletion, causing the removal of the strand betaF. This strand is one of the seven strands that compose the core beta-sheet of the Rossman fold dinucleotide-binding motif characteristic of the short chain dehydrogenase/reductase (SDR) family members. However, the fact that the substrate binding cleft residues are retained and the existence of a shared variant between human and dog suggest that it might be functional. Molecular adaptation analyses across eutherian mammal orthologues revealed the existence of sites under positive selection, some of which being localized in the substrate-binding cleft and in the insertion 1 region on loop D (an important region for the Abeta-binding to the enzyme). Interestingly, a higher than expected number of nonsynonymous substitutions were observed between human/chimpanzee and orangutan, with six out of the seven amino acid replacements being under molecular adaptation (including three in loop D and one in the substrate binding loop). CONCLUSION: Our study revealed that HADH2 genes maintained a reasonable conserved organization across a large evolutionary distance. The conserved noncoding regions identified among mammals and between pufferfishes, the evidence of an alternative splicing variant conserved between human and dog, and the detection of positive selection across eutherian mammals, may be of importance for further research on ABAD/HSD10 function and its implication in the Alzheimer's disease.

17-Hydroxysteroid Dehydrogenases↗

PubMed2006

Biological evaluation, chelation, and molecular modeling studies of novel metal-chelating inhibitors of NF-kappaB-DNA binding: structure activity relationships.

Previously, we have reported that aurintricarboxylic acid (ATA) is one of the most potent inhibitors of the DNA binding of transcription factor NF-kappaB. We now report the NF-kappaB-DNA binding inhibitory activity of ATA analogues. An electrophoretic mobility shift assay has shown that bromopyrogallol red (BPR) is the most effective inhibitor of NF-kappaB-DNA binding among the studied analogues. The molecular modeling studies showed that BPR makes a strong network of hydrogen bonds with the DNA-binding region of the p50 subunit of NF-kappaB and has electronegative potential on its peripheral surface. Because zinc has been reported to influence the DNA binding of NF-kappaB, the interaction of these analogues with zinc was studied. Chemical speciation and formation-constant studies showed that BPR forms the most stable 1:1 complex with zinc. BPR has also been found to be the most potent antioxidant among the studied analogues.

Antiviral Agents↗

PubMed2006

Unraveling the importance of protein-protein interaction: application of a computational alanine-scanning mutagenesis to the study of the IgG1 streptococcal protein G (C2 fragment) complex.

Alanine-scanning mutagenesis of protein-protein interfacial residues is a very important process for rational drug design. In this study, we have used the improved MM-PBSA approach that combining molecular mechanics and continuum solvent permits one to calculate the free energy differences through alanine mutation. To identify the binding determinants of the complex formed between the IgG1 (immunoglobulin-binding protein G) and protein G, we have extended the experimental alanine scanning mutagenesis study to both proteins of this complex and, therefore, to all interfacial residues of this binding complex. As a result, we present new residues that can be characterized as warm spots and, therefore, are important for complex formation. We have further increased the understanding of the functionality of this improved computational alanine-scanning mutagenesis approach testing its sensitivity to a protein-protein complex with an interface made up of residues mainly polar. In this study, we also have improved the method for the detection of an important amino acid residue that frequently constitutes a hot spot--tryptophan.

Alanine↗

PubMed2006

Mechanism of a soluble fumarate reductase from Shewanella frigidimarina: a theoretical study.

The mechanism of a unique fumarate reductase is explored using the hybrid density functional B3LYP method. The calculations show a two-step mechanism, initiated with a hydride transfer from FAD (flavin adenine dinucleotide) to fumarate, followed by a proton shift from Arg402. The rate-limiting process is assigned to the hydride transfer, and the energetics are consistent with experimental data. It is shown that the enzyme is essential to correctly position the substrate in the active site, stabilizing its extremely anionic character.

Models, Molecular↗

PubMed2006

Theoretical study of the unusual protonation properties of the active site cysteines in thioredoxin.

The thiol/disulfide oxidoreductases of the thioredoxin family have, in the active site, two cysteines that can be in a reduced or an oxidized form. One of the cysteines in the reduced state is deprotonated, and it is called nucleophilic cysteine. The pK(a) of this cysteine is different from that of a normal cysteine and varies widely among the different enzymes of this family. However, the factors responsible for the different degrees of stabilization of nucleophilic cysteine thiolate are not fully understood. Here, we have studied the well-known hypothesis of proton sharing between the active site thiols by performing a linear transit scan for the transfer of the proton between the active site cysteines. We used a two-layered (DFT/MM) ONIOM formalism, with the active site region treated at the B3LYP/6-31+G(d) level and the remains of the protein treated with the Amber Parm94 force field. The solvation free energy was accounted for with a continuum solvent model, by solving the Poisson-Boltzmann equation using the program Delphi. We have obtained excellent agreement with the experimental data available in the literature. Besides refuting the proton sharing hypothesis, our results include a value of 14.0 for the pK(a) of the buried cysteine, a quantity that has not been possible to obtain experimentally but which has been proven to be higher than 11. Additionally, this study also provides detailed information on the very interesting and so far unknown fact that the contribution of the enzymatic structure (8.3 kcal/mol) prevails in relation to that of the solvent (0.60 kcal/mol) concerning the differential stabilization of the active site thiolates.

Binding Sites↗

PubMed2006

Determination of the DeltapKa between the active site cysteines of thioredoxin and DsbA.

Thioredoxin superfamily members share a considerable degree of structural similarity, with a conserved CX(i)X(j)C motif at the active site, where C stand for two cysteines that alternate between a reduced thiol and oxidized disulfide states, and X(i)and X(j) are two amino acids different in each family member. Despite these similarities, they display very different redox potentials and pKas for the active site dithiol, and fulfill different physiological roles. Thioredoxin, for example, promotes the reduction of disulfide bonds, while DsbA promotes their oxidation in prokaryotic cells. The factors that promote these differences are still not fully understood. However, it is generally accepted that the different stabilities of the redox active disulfide bond depends on the degree of stabilization, in the reduced state, of the thiolate of one of the active site cysteines (nucleophilic cysteine). In this work we have used QM/MM methods to compare and characterize the active site dithiols of both enzymes, and to shed some light on the structural features responsible for the large differences in pKa and redox potential between two homologous enzymes, thioredoxin and DsbA. We have also analyzed the main factors pointed out in the literature as responsible for their different properties. We obtained the value of 4.5 for pKa difference (DeltapKa) between the nucleophilic cysteines of both enzymes, which is in excellent agreement with most of the experimental values. Additionally, we found that the principal differentiating factor responsible for this observed DeltapKa are the alpha2-alpha helices, which greatly contribute to the mentioned value, by stabilizing the DsbA thiolate in a much greater extend than the thioredoxin thiolate. A double mutation of the conserved residues Asp26 and Lys57, in thioredoxin, and Glu24 Lys58, in DsbA, by alanines did not change the DeltapKa value; this supports the hypothesis that these residues are not involved in the differentiation of the properties of the active centre dithiol. However, we found out that these residues are important for the stabilization of the nucleophilic thiolate. The X(i) and X(j) residues also do not seem to promote the stabilization of the thiolates. In fact, the corresponding double alanine mutants are more stable than the wild-type enzymes. However, these residues are involved in the differentiation between thioredoxin and DsbA, stabilizing the DsbA thiolate by a larger extent than the thioredoxin thiolate.

Amino Acid Substitution↗

PubMed2006

Molecular insights into the mechanisms of HIV-1 reverse transcriptase resistance to nucleoside analogs.

The causative agent of acquired immunodeficiency syndrome, HIV-1, depends on one of its enzymes, reverse transcriptase, to copy its single stranded RNA genome into a double stranded DNA nucleic acid suitable for integration in the host cell genome. In the last two decades, the advances in the knowledge of the kinetic mechanism of reverse transcription and in the determination of the crystallographic structures for the complexes of the enzyme with substrates and products were huge. However, all of this knowledge resulted in the design of RT inhibitors for which the virus, after a short period of exposure, becomes less susceptible, due to the development of resistance. The development of resistance is caused by the high frequency of viral mutation and the toxicity of those same drugs. Therefore, a closer look at all the available information might shed some light into this subject and help to develop new strategies to overcome the lack of long term clinical efficiency of these drugs. Here, we present a critical atomic level study of all the mutations that have been detected and reported so far, as a reaction of the enzyme to counteract the action of the inhibitors.

Drug Resistance, Viral↗

PubMed2005

Structural basis for the GSK-3beta binding affinity and selectivity against CDK-2 of 1-(4-aminofurazan-3yl)-5-dialkylaminomethyl-1H-[1,2,3] triazole-4-carboxylic acid derivatives.

A novel structural class of glycogen synthase kinase-3beta inhibitors is modeled using quantum mechanics, automated docking, and molecular dynamics simulations. The proposed binding modes identify important hydrogen bonds and salt-bridges with the ATP-binding pocket of the kinase. The modeled complexes justify the observed structure-activity relationships and provide a structural basis for the high selectivity of these inhibitors against cyclin dependent kinase-2.

Adenosine Triphosphate↗

PubMed2005

Molecular dynamics simulations of angiotensin II in aqueous and dimethyl sulfoxide environments.

Angiotensin II (Ang II) is an octapeptidic hormone, which plays an important role in the mechanisms of blood pressure control. In this work, extensive molecular dynamics (MD) simulations have been carried out on this peptide, both in aqueous and in dimethyl sulfoxide (DMSO) environments. Experimentally proposed models for the structure of angiotensin II in both environments are not consensual and the results obtained have provided some further insight about the structural properties of this hormone. In these simulations, the N-terminus of Ang II in the aqueous environment has been associated with a considerable larger flexibility than the correspondent C-terminus, but this was not found in the case of the DMSO environment. This is consistent with the assumption that the biological activity of Ang II is associated with its C-terminal residues embedded in a hydrophobic environment of its AT1 receptor. Other features detected in DMSO environment were an H(His6 imidazole)-O(Phe8 carboxylate) hydrogen bond and a salt-bridge structure involving the Asp1 and Arg2 side chains. An additional important conformational feature is the spatial proximity between Tyr4 and His6 in both water and DMSO environments. This molecular feature may trigger the interest for the synthetic chemists to apply rational design for the synthesis of novel AT1 antagonists.

Angiotensin II↗

PubMed2005

Molecular recognition of 15-deoxy-delta(12,14)-prostaglandin J2 by nuclear factor-kappa B and other cellular proteins.

15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), a dehydration product of prostaglandin D2, is an important pharmacological molecule, which with the virtue of its electrophilicity, has been reported to covalently modify some cellular proteins (such as nuclear factor-kappa B (NF-kappaB), AP-1, p53, and thioredoxin) and elicit its physiological effects. The aim of the present computational study is to understand the role molecular recognition plays in the association of 15d-PGJ2 with NF-kappaB and other proteins. Another aim is to characterize whether p53 is a direct target for covalent modification by 15d-PGJ2. A docking strategy is applied along with calculation of ab initio electrostatic potential maps to analyze the mode of binding of prostaglandin molecule with critical cysteine-containing sites in each protein. The results provide identification of important sites in the target proteins, which provide recognition and stability to the prostaglandin molecule. Fit of shape and complementarity of electrostatic interactions are derived as significant determinants of molecular recognition of 15d-PGJ2. Further, comparative results indicate that p53 protein may also be a target for direct modification by 15d-PGJ2. The molecular models obtained should allow the rational design of more specific analogs of 15d-PGJ2.

Binding Sites↗

PubMed2005

Similarities and differences in the thioredoxin superfamily.

There is growing interest in the proteins involved in protein folding. This is mainly due to the large number of human diseases related to defects in folding, which include cystic fibrosis, Alzheimer's and cancer. However, equally important as the oxidation and concomitant formation of disulfide bridges of the extracellular or secretory proteins is the reduction and maintenance in the reduced state of the proteins within the cell. Interestingly, the proteins that are responsible for maintenance of the reduced state belong to the same superfamily as those responsible for the formation of disulfide bridges: all are members of the thioredoxin superfamily. In this article, we highlight the main features of those thioredoxin-like proteins directly involved in the redox reactions. We describe their biological functions, cytoplasmic location, mechanisms of action, structures and active site features, and discuss the principal hypotheses concerning origins of the different reduction potentials and unusual pK(a)'s of the catalytic residues.

Amino Acid Motifs↗

PubMed2005

Inhibitory activity of polyhydroxycarboxylate chelators against recombinant NF-kappaB p50 protein-DNA binding.

The inhibitory effect of 7,8-dihydroxy-4-methylcoumarin (7,8-DHMC), 5,7-dihydroxy-4-methylcoumarin (5,7-DHMC), and gallic acid on the DNA binding of recombinant p50 protein and their interaction with zinc ion were studied. Electrophoretic mobility shift assay (EMSA) using p50 and biotin labeled DNA has shown that gallic acid is more effective than the dihydroxycoumarins in inhibiting the p50-DNA binding. Molecular modeling studies suggest an explanation for these observations. Effect of the addition of zinc after p50-DNA-binding inhibition by gallic acid was also studied. Chemical speciation and formation constant studies show that gallic acid forms a more stable 1:1 complex with zinc ion in comparison to the dihydroxycoumarins.

Cations, Divalent↗

PubMed2005

Overview of ribonucleotide reductase inhibitors: an appealing target in anti-tumour therapy.

This review provides up-to-date information on the inhibition of ribonucleotide reductase (RNR), the enzyme that catalyses the reduction of ribonucleotides into deoxyribonucleotides. Taking in account that DNA replication and repair are essential mechanisms for cell integrity and are dependent on the availability of deoxyribonucleotides, many researchers are giving special attention to this enzyme, since it is an attractive target to treat several diseases of our time specially cancer. This investment has already given some benefits since some of these inhibitors show potent chemotherapeutic efficacy against a wide range of tumours such as non-small cell lung cancer, adenocarcinoma of pancreas, bladder cancer, leukaemia and some solid tumours. In fact a few of them have already been approved for the clinical treatment of some kinds of cancer. All aspects of RNR inhibition and corresponding inhibitors are the subjects of this review. The inhibitors are divided in three main groups: translation inhibitors, which unable the formation of the enzyme; dimerization inhibitors that prevent the complexation of the two RNR subunits (R1 and R2); and catalytic inhibitors that inactivate subunit R1 and/or subunit R2, leading to RNR inactivity. In this last group special focus will be addressed to substrate analogues.

Animals↗