Biomedical subjects

M Karplus

Publications and source records attributed to M Karplus.

At least 19 recordsLinked to original sources

PubMed2005

Glycogen phosphorylase inhibitors: a free energy perturbation analysis of glucopyranose spirohydantoin analogues.

GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.

Computer Simulation↗

PubMed2005

Molecular dynamics and protein function.

A fundamental appreciation for how biological macromolecules work requires knowledge of structure and dynamics. Molecular dynamics simulations provide powerful tools for the exploration of the conformational energy landscape accessible to these molecules, and the rapid increase in computational power coupled with improvements in methodology makes this an exciting time for the application of simulation to structural biology. In this Perspective we survey two areas, protein folding and enzymatic catalysis, in which simulations have contributed to a general understanding of mechanism. We also describe results for the F(1) ATPase molecular motor and the Src family of signaling proteins as examples of applications of simulations to specific biological systems.

Animals↗

PubMed2003

The missing link between thermodynamics and structure in F1-ATPase.

F(1)F(o)-ATP synthase is the enzyme responsible for most of the ATP synthesis in living systems. The catalytic domain F(1) of the F(1)F(o) complex, F(1)-ATPase, has the ability to hydrolyze ATP. A fundamental problem in the development of a detailed mechanism for this enzyme is that it has not been possible to determine experimentally the relation between the ligand binding affinities measured in solution and the different conformations of the catalytic beta subunits (beta(TP), beta(DP), beta(E)) observed in the crystal structures of the mitochondrial enzyme, MF(1). Using free energy difference simulations for the hydrolysis reaction ATP+H(2)O --> ADP+P(i) in the beta(TP) and beta(DP) sites and unisite hydrolysis data, we are able to identify beta(TP) as the "tight" (K(D) = 10(-12) M, MF(1)) binding site for ATP and beta(DP) as the "loose" site. An energy decomposition analysis demonstrates how certain residues, some of which have been shown to be important in catalysis, modulate the free energy of the hydrolysis reaction in the beta(TP) and beta(DP) sites, even though their structures are very similar. Combined with the recently published simulations of the rotation cycle of F(1)-ATPase, the present results make possible a consistent description of the binding change mechanism of F(1)-ATPase at an atomic level of detail.

Adenosine Diphosphate↗

PubMed2003

Protein-ligand binding free energy estimation using molecular mechanics and continuum electrostatics. Application to HIV-1 protease inhibitors.

A method is proposed for the estimation of absolute binding free energy of interaction between proteins and ligands. Conformational sampling of the protein-ligand complex is performed by molecular dynamics (MD) in vacuo and the solvent effect is calculated a posteriori by solving the Poisson or the Poisson-Boltzmann equation for selected frames of the trajectory. The binding free energy is written as a linear combination of the buried surface upon complexation, SASbur, the electrostatic interaction energy between the ligand and the protein, Eelec, and the difference of the solvation free energies of the complex and the isolated ligand and protein, deltaGsolv. The method uses the buried surface upon complexation to account for the non-polar contribution to the binding free energy because it is less sensitive to the details of the structure than the van der Waals interaction energy. The parameters of the method are developed for a training set of 16 HIV-1 protease-inhibitor complexes of known 3D structure. A correlation coefficient of 0.91 was obtained with an unsigned mean error of 0.8 kcal/mol. When applied to a set of 25 HIV-1 protease-inhibitor complexes of unknown 3D structures, the method provides a satisfactory correlation between the calculated binding free energy and the experimental pIC5o without reparametrization.

HIV-1↗

PubMed2003

Successful control of an Acinetobacter baumannii outbreak in a neonatal intensive care unit.

We describe an outbreak of Acinetobacter baumannii in a neonatal intensive care unit (NICU), and our investigation to determine the source and mode of transmission and identify the population at risk. A case (infected infant) was defined as a patient hospitalized in the NICU during the outbreak period, with clinical signs of sepsis and isolation of A. baumannii. In colonized infants, A. baumannii was isolated from body surfaces without signs of infection. Infected infants were separated and treated by a different medical team. Cultures were taken from working surfaces and along the infant's admission passage from the delivery room to the NICU. The outbreak strain was identified by pulsed-field gel electrophoresis (PFGE). Nine cases and eight colonized infants met the definition criteria. Cases were younger than colonized infants with regard to gestational age and age of diagnosis and had lower birthweights (P<0.01). The outbreak strain was only isolated from hygroscopic bandages used on skin under the ventilation tube and umbilical catheters. Discontinuing the use of the bandages put an end to the outbreak. We conclude that a rapid and thorough investigation of the environment during an outbreak of A. baumannii is essential to finding the source of the infection, and that hygroscopic bandages may be a source of such outbreaks.

Acinetobacter Infections↗

PubMed2003

Neonatologists are using much less dexamethasone.

Two historical cohorts (1993-1994 and 2001) of preterm infants ventilated for respiratory distress syndrome were compared. Dexamethasone administration fell from 22% to 6%. Chronic lung disease in survivors rose slightly from 13% to 17%, and mortality fell from 21% to 15% (other causes). The effect of restriction of dexamethasone use on chronic lung disease and mortality remains to be seen.

Birth Weight↗

PubMed2002

Small-world view of the amino acids that play a key role in protein folding.

We use geometrical considerations to provide a different perspective on the fact that a few selected amino acids, the so-called "key residues," act as nucleation centers for protein folding. By constructing graphs corresponding to protein structures we show that they have the "small-world" feature of having a limited set of vertices with large connectivity. These vertices correspond to the key residues that play the role of "hubs" in the network of interactions that stabilize the structure of the transition state.

Amino Acids↗

PubMed2002

Relation between sequence and structure of HIV-1 protease inhibitor complexes: a model system for the analysis of protein flexibility.

The flexibility of different regions of HIV-1 protease was examined by using a database consisting of 73 X-ray structures that differ in terms of sequence, ligands or both. The root-mean-square differences of the backbone for the set of structures were shown to have the same variation with residue number as those obtained from molecular dynamics simulations, normal mode analyses and X-ray B-factors. This supports the idea that observed structural changes provide a measure of the inherent flexibility of the protein, although specific interactions between the protease and the ligand play a secondary role. The results suggest that the potential energy surface of the HIV-1 protease is characterized by many local minima with small energetic differences, some of which are sampled by the different X-ray structures of the HIV-1 protease complexes. Interdomain correlated motions were calculated from the structural fluctuations and the results were also in agreement with molecular dynamics simulations and normal mode analyses. Implications of the results for the drug-resistance engendered by mutations are discussed briefly.

Amino Acid Substitution↗

PubMed2001

Use of MCSS to design small targeted libraries: application to picornavirus ligands.

Computational methods were used to design structure-based combinatorial libraries of antipicornaviral capsid-binding ligands. The multiple copy simultaneous search (MCSS) program was employed to calculate functionality maps for many diverse functional groups for both the poliovirus and rhinovirus capsid structures in the region of the known drug binding pocket. Based on the results of the MCSS calculations, small combinatorial libraries consisting of 10s or 100s of three-monomer compounds were designed and synthesized. Ligand binding was demonstrated by a noncell-based mass spectrometric assay, a functional immuno-precipitation assay, and crystallographic analysis of the complexes of the virus with two of the candidate ligands. The P1/Mahoney poliovirus strain was used in the experimental studies. A comparison showed that the MCSS calculations had correctly identified the observed binding site for all three monomer units in one ligand and for two out of three in the other ligand. The correct central monomer position in the second ligand was reproduced in calculations in which the several key residues lining the pocket were allowed to move. This study validates the computational methodology. It also illustrates that subtle changes in protein structure can lead to differences in docking results and points to the importance of including target flexibility, as well as ligand flexibility, in the design process.

Benzimidazoles↗

PubMed2001

Forces and energetics of hapten-antibody dissociation: a biased molecular dynamics simulation study.

The unbinding of fluorescein from the single-chain Fv fragment of the 4D5Flu antibody is investigated by biased molecular dynamics with an implicit solvation model. To obtain statistically meaningful results, a large number of unbinding trajectories are calculated; they involve a total simulation time of more than 200 ns. Simulations are carried out with a time-dependent perturbation and in the presence of a constant force. The two techniques, which provide complementary information, induce unbinding by favoring an increase in the distance between the ligand and the antibody. This distance is an appropriate progress variable for the dissociation reaction and permits direct comparison of the unbinding forces in the simulations with data from atomic force microscopy (AFM). The time-dependent perturbation generates unfolding pathways that are close to equilibrium and can be used to reconstruct the mean force; i.e. the derivative of the potential of mean force, along the reaction coordinate. This is supported by an analysis of the overall unbinding profile and the magnitude of the mean force, which are similar to those of the unbinding force (i.e. the external force due to the time-dependent perturbation) averaged over several unbinding events. The multiple simulations show that unbinding proceeds along a rather well-defined pathway for a broad range of effective pulling speeds. Initially, there is a distortion of the protein localized in the C-terminal region followed by the fluorescein exit from the binding site. This occurs in steps that involve breaking of specific electrostatic and van der Waals interactions. It appears that the simulations do not explore the same barriers as those measured in the AFM experiments because of the much higher unfolding speed in the former. The dependence of the force on the logarithm of the loading rate is linear and the slope is higher than in the AFM, in agreement with experiment in other systems, where different slopes were observed for different regimes. Based on the unbinding events, mutations in the 4D5Flu antigen binding site are predicted to result in significant changes in the unbinding force.

Animals↗

PubMed2001

Solvent effects on the reaction coordinate of the hydrolysis of phosphates and sulfates: application of Hammond and anti-Hammond postulates to understand hydrolysis in solution.

The mechanism of the alkaline hydrolysis of phosphate and sulfate esters is of great interest. Ab initio quantum mechanical calculations and dielectric continuum methods are used to investigate the effect of the solvent on the associative/dissociative and the in-line/sideways character of the hydrolysis reaction of ethylene sulfate (ES) and ethylene phosphate (EP(-)), and their acyclic counterparts, dimethyl sulfate (DMS) and dimethyl phosphate (DMP(-)). The gas-phase reaction coordinates are determined by Hartree-Fock and density functional theory. For ES, the reaction coordinate in solution is determined; for the other three reactions only the transition state in solution is obtained. The alterations in the reaction induced by solvent are interpreted by use of the Hammond and anti-Hammond postulates.

Ethylenes↗

PubMed2001

Uracil-DNA glycosylase acts by substrate autocatalysis.

In humans, uracil appears in DNA at the rate of several hundred bases per cell each day as a result of misincorporation of deoxyuridine (dU) or deamination of cytosine. Four enzymes that catalyse the hydrolysis of the glycosylic bond of dU in DNA to yield an apyridiminic site as the first step in base excision repair have been identified in the human genome. The most efficient and well characterized of these uracil-DNA glycosylases is UDG (also known as UNG and present in almost all known organisms), which excises U from single- or double-stranded DNA and is associated with DNA replication forks. We used a hybrid quantum-mechanical/molecular-mechanical (QM/MM) approach to determine the mechanism of catalysis by UDG. In contrast to the concerted associative mechanism proposed initially, we show here that the reaction proceeds in a stepwise dissociative manner. Cleavage of the glycosylic bond yields an intermediate comprising an oxocarbenium cation and a uracilate anion. Subsequent attack by a water molecule and transfer of a proton to D145 result in the products. Surprisingly, the primary contribution to lowering the activation energy comes from the substrate, rather than from the enzyme. This 'autocatalysis' derives from the burial and positioning of four phosphate groups that stabilize the rate-determining transition state. The importance of these phosphates explains the residual activity observed for mutants that lack key residues. A corresponding catalytic mechanism could apply to the DNA glycosylases TDG and SMUG1, which belong to the same structural superfamily as UDG.

Catalysis↗

PubMed2001

The energetics of off-rotamer protein side-chain conformations.

Non-rotameric ("off-rotamer") conformations are commonly observed for the side-chains of protein crystal structures. This study examines whether such conformations are real or artifactual by comparing the energetics of on and off-rotamer side-chain conformations calculated with the CHARMM energy function. Energy-based predictions of side-chain orientation are carried out by rigid-geometry mapping in the presence of the fixed protein environment for 1709 non-polar side-chains in 24 proteins for which high-resolution (2.0 A or better) structures are available. For on-rotamer conformations, 97.6 % are correctly predicted; i.e. they correspond to the absolute minima of their local side-chain energy maps (generally to within 10 degrees or less). By contrast, for the observed off-rotamer side-chain conformations, 63.8 % are predicted correctly. This difference is statistically significant (P<0.001) and suggests that while most of the observed off-rotamer conformations are real, many of the erroneously predicted ones are likely to be artifacts of the X-ray refinements. Probabilities for off-rotamer conformations of the non-polar side-chains are calculated to be 5.0-6.1 % by adaptive umbrella-sampled molecular dynamics trajectories of individual amino acid residues in vacuum and in the presence of an average protein or aqueous dielectric environment. These results correspond closely to the 5.7 % off-rotamer fraction predicted by the rigid-geometry mapping studies. Since these values are about one-half of the 10.2 % off-rotamer fraction observed in the X-ray structures, they support the conclusion that many of the latter are artifacts. In both the rigid-geometry mapping and the molecular dynamics studies, the discrepancies between the predicted and observed fractions of off-rotamer conformations are largest for leucine residues (approximately 6 % versus 16.6 %). The simulations for the isolated amino acid residues indicate that the real off-rotamer frequency of 5-6 % is consistent with the internal side-chain and local side-chain-backbone energetics and does not originate from shifts due to the protein. The present results suggest that energy-based rotation maps can be used to find side-chain positional artifacts that appear in crystal structures based on refinements in the 2 A resolution range.

Amino Acids↗

PubMed2001

Crystal structures of two closely related but antigenically distinct HLA-A2/melanocyte-melanoma tumor-antigen peptide complexes.

We have determined high-resolution crystal structures of the complexes of HLA-A2 molecules with two modified immunodominant peptides from the melanoma tumor-associated protein Melan-A/Melanoma Ag recognized by T cells-1. The two peptides, a decamer and nonamer with overlapping sequences (ELAGIGILTV and ALGIGILTV), are modified in the second residue to increase their affinity for HLA-A2. The modified decamer is more immunogenic than the natural peptide and a candidate for peptide-based melanoma immunotherapy. The crystal structures at 1.8 and 2.15 A resolution define the differences in binding modes of the modified peptides, including different clusters of water molecules that appear to stabilize the peptide-HLA interaction. The structures suggest both how the wild-type peptides would bind and how three categories of cytotoxic T lymphocytes with differing fine specificity might recognize the two peptides.

Amino Acid Sequence↗

PubMed2001

Substrate conformational transitions in the active site of chorismate mutase: their role in the catalytic mechanism.

Chorismate mutase acts at the first branch-point of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. The results of molecular dynamics simulations of the substrate in solution and in the active site of chorismate mutase are reported. Two nonreactive conformers of chorismate are found to be more stable than the reactive pseudodiaxial chair conformer in solution. It is shown by QM/MM molecular dynamics simulations, which take into account the motions of the enzyme, that when these inactive conformers are bound to the active site, they are rapidly converted to the reactive chair conformer. This result suggests that one contribution of the enzyme is to bind the more prevalent nonreactive conformers and transform them into the active form in a step before the chemical reaction. The motion of the reactive chair conformer in the active site calculated by using the QM/MM potential generates transient structures that are closer to the transition state than is the stable CHAIR conformer.

Binding Sites↗

PubMed2001

Triosephosphate isomerase: a theoretical comparison of alternative pathways.

Three mechanisms proposed for the triosephosphate isomerase (TIM) catalyzed reactions were studied with the QM/MM approach using B3LYP/6-31+G(d,p) as the QM method. The two pathways that involve an enediol species were found to give similar values for the barriers and the calculated rates are in satisfactory agreement with experiment. By contrast, the mechanism that involves intramolecular proton transfer in the enediolate was found to be energetically unfavorable due to electrostatic interactions with His 95, a conserved residue in TIM from different organisms. A perturbation analysis was used to determine the residues that make the major contribution to catalysis.

Kinetics↗

PubMed2001

Binding free energies and free energy components from molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition by aspartyl-tRNA synthetase.

Specific amino acid binding by aminoacyl-tRNA synthetases (aaRS) is necessary for correct translation of the genetic code. Engineering a modified specificity into aminoacyl-tRNA synthetases has been proposed as a means to incorporate artificial amino acid residues into proteins in vivo. In a previous paper, the binding to aspartyl-tRNA synthetase of the substrate Asp and the analogue Asn were compared by molecular dynamics free energy simulations. Molecular dynamics combined with Poisson-Boltzmann free energy calculations represent a less expensive approach, suitable for examining multiple active site mutations in an engineering effort. Here, Poisson-Boltzmann free energy calculations for aspartyl-tRNA synthetase are first validated by their ability to reproduce selected molecular dynamics binding free energy differences, then used to examine the possibility of Asn binding to native and mutant aspartyl-tRNA synthetase. A component analysis of the Poisson-Boltzmann free energies is employed to identify specific interactions that determine the binding affinities. The combined use of molecular dynamics free energy simulations to study one binding process thoroughly, followed by molecular dynamics and Poisson-Boltzmann free energy calculations to study a series of related ligands or mutations is proposed as a paradigm for protein or ligand design. The binding of Asn in an alternate, "head-to-tail" orientation observed in the homologous asparagine synthetase is analyzed, and found to be more stable than the "Asp-like" orientation studied earlier. The new orientation is probably unsuitable for catalysis. A conserved active site lysine (Lys198 in Escherichia coli) that recognizes the Asp side-chain is changed to a leucine residue, found at the corresponding position in asparaginyl-tRNA synthetase. It is interesting that the binding of Asp is calculated to increase slightly (rather than to decrease), while that of Asn is calculated, as expected, to increase strongly, to the same level as Asp binding. Insight into the origin of these changes is provided by the component analyses. The double mutation (K198L,D233E) has a similar effect, while the triple mutation (K198L,Q199E,D233E) reduces Asp binding strongly. No binding measurements are available, but the three mutants are known to have no ability to adenylate Asn, despite the "Asp-like" binding affinities calculated here. In molecular dynamics simulations of all three mutants, the Asn ligand backbone shifts by 1-2 A compared to the experimental Asp:AspRS complex, and significant side-chain rearrangements occur around the pocket. These could reduce the ATP binding constant and/or the adenylation reaction rate, explaining the lack of catalytic activity in these complexes. Finally, Asn binding to AspRS with neutral K198 or charged H449 is considered, and shown to be less favorable than with the charged K198 and neutral H449 used in the analysis.

Amino Acid Substitution↗

PubMed2001

Exploration of partially unfolded states of human alpha-lactalbumin by molecular dynamics simulation.

Molecular dynamics simulations are used to probe the properties of non-native states of the protein human alpha-lactalbumin (human alpha-LA) with a detailed atomistic model in an implicit aqueous solvent environment. To sample the conformational space, a biasing force is introduced that increases the radius of gyration relative to the native state and generates a large number of low-energy conformers that differ in terms of their root-mean-square deviation, for a given radius of gyration. The resulting structures are relaxed by unbiased simulations and used as models of the molten globule and partly denatured states of human alpha-LA, based on measured radii of gyration obtained from nuclear magnetic resonance experiments. The ensembles of structures agree in their overall properties with experimental data available for the human alpha-LA molten globule and its more denatured states. In particular, the simulation results show that the native-like fold of the alpha-domain is preserved in the molten globule. Further, a considerable proportion of the antiparallel beta-strand in the beta-domain are present. This indicates that the lack of hydrogen exchange protection found experimentally for the beta-domain is due to rearrangement of the beta-sheet involving transient populations of non-native beta-structures. The simulations also provide details concerning the ensemble of structures that contribute as the molten globule unfolds and shows, in accord with experimental data, that unfolding is not cooperative; i.e. the various structural elements do not unfold simultaneously.

Binding Sites↗