A conformational transition in the myosin VI converter contributes to the variable step size.

Myosin VI (MVI) is a dimeric molecular motor that translocates backwards on actin filaments with a surprisingly large and variable step size, given its short lever arm. A recent x-ray structure of MVI indicates that the large step size can be explained in part by a novel conformation of the converter subdomain in the prepowerstroke state, in which a 53-residue insert, unique to MVI, reorients the lever arm nearly parallel to the actin filament. To determine whether the existence of the novel converter conformation could contribute to the step-size variability, we used a path-based free-energy simulation tool, the string method, to show that there is a small free-energy difference between the novel converter conformation and the conventional conformation found in other myosins. This result suggests that MVI can bind to actin with the converter in either conformation. Models of MVI/MV chimeric dimers show that the variability in the tilting angle of the lever arm that results from the two converter conformations can lead to step-size variations of ∼12 nm. These variations, in combination with other proposed mechanisms, could explain the experimentally determined step-size variability of ∼25 nm for wild-type MVI. Mutations to test the findings by experiment are suggested.

Calculation of free-energy differences by confinement simulations. Application to peptide conformers.

Conformational free-energy differences are key quantities for understanding important phenomena in molecular biology that involve large structural changes of macromolecules. In this paper, an improved version of the confinement approach, which is based on earlier developments, is used to determine the free energy of individual molecular states by progressively restraining the corresponding molecular structures to pure harmonic basins, whose absolute free energy can be computed by normal-mode analysis. The method is used to calculate the free-energy difference between two conformational states of the alanine dipeptide in vacuo, and of the beta-hairpin from protein G with an implicit solvation model. In all cases, the confinement results are in excellent agreement with the ones obtained from converged equilibrium molecular dynamics simulations, which have a much larger computational cost. The systematic and statistical errors of the results are evaluated and the origin of the errors is identified. The sensitivity of the calculated free-energy differences to structure-based definitions of the molecular states is discussed. A variant of the method, which closes the thermodynamic cycle by a quasi-harmonic rather than harmonic analysis, is introduced. The latter is proposed for possible use with explicit solvent simulations.

CHARMM: the biomolecular simulation program.

CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates, and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. The CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This article provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM article in 1983.

Allosteric communication in myosin V: from small conformational changes to large directed movements.

The rigor to post-rigor transition in myosin, a consequence of ATP binding, plays an essential role in the Lymn-Taylor functional cycle because it results in the dissociation of the actomyosin complex after the powerstroke. On the basis of the X-ray structures of myosin V, we have developed a new normal mode superposition model for the transition path between the two states. Rigid-body motions of the various subdomains and specific residues at the subdomain interfaces are key elements in the transition. The allosteric communication between the nucleotide binding site and the U50/L50 cleft is shown to result from local changes due to ATP binding, which induce large amplitude motions that are encoded in the structure of the protein. The triggering event is the change in the interaction of switch I and the P-loop, which is stabilized by ATP binding. The motion of switch I, which is a relatively rigid element of the U50 subdomain, leads directly to a partial opening of the U50/L50 cleft; the latter is expected to weaken the binding of myosin to actin. The calculated transition path demonstrates the nature of the subdomain coupling and offers an explanation for the mutual exclusion of ATP and actin binding. The mechanism of the uncoupling of the converter from the motor head, an essential part of the transition, is elucidated. The origin of the partial untwisting of the central beta-sheet in the rigor to post-rigor transition is described.

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.

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.

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.

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.

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.

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.

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.

Effect of position changing on bilirubin levels during phototherapy.

OBJECTIVE: Turning of infants during phototherapy for hyperbilirubinemia is practiced in many nurseries. However, there is little research evidence in support of this practice. This study examined the effect of turning on serum total bilirubin concentration and on the duration of phototherapy. STUDY DESIGN: We first conducted a pilot study in term infants requiring phototherapy using transcutaneous bilirubinometry in order to determine the time required to clear the skin of bilirubin. This "blanching time" was found to be approximately 150 minutes. We then conducted a randomized study comparing turning the baby during phototherapy with care in the supine position only. RESULTS: Thirty term infants were enrolled in the study (turned - 14; supine - 16). No differences were found between the groups in baseline data, such as birth weight, gestational age, age at start of phototherapy, or type of feeds. Infants in the supine group showed a significantly larger drop in serum total bilirubin concentration and required a shorter duration of phototherapy. CONCLUSION: We conclude that infants should be nursed supine during phototherapy. Based on these results, we propose a modification to the traditional model of bilirubin kinetics during phototherapy.

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.

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.

Evaluation of designed ligands by a multiple screening method: application to glycogen phosphorylase inhibitors constructed with a variety of approaches.

Glycogen phosphorylase (GP) is an important enzyme that regulates blood glucose level and a key therapeutic target for the treatment of type II diabetes. In this study, a number of potential GP inhibitors are designed with a variety of computational approaches. They include the applications of MCSS, LUDI and CoMFA to identify additional fragments that can be attached to existing lead molecules; the use of 2D and 3D similarity-based QSAR models (HQSAR and SMGNN) and of the LUDI program to identify novel molecules that may bind to the glucose binding site. The designed ligands are evaluated by a multiple screening method, which is a combination of commercial and in-house ligand-receptor binding affinity prediction programs used in a previous study (So and Karplus, J. Comp.-Aid. Mol. Des., 13 (1999), 243-258). Each method is used at an appropriate point in the screening, as determined by both the accuracy of the calculations and the computational cost. A comparison of the strengths and weaknesses of the ligand design approaches is made.

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.

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.

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.

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.

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.