Molecular dynamics simulations of trehalose as a 'dynamic reducer' for solvent water molecules in the hydration shell

Systematic computational work for a series of 13 disaccharides was performed to provide an atomic-level insight of unique biochemical role of the alpha,alpha-(1-->1)-linked glucopyranoside dimer over the other glycosidically linked sugars. Superior osmotic and cryoprotective abilities of trehalose were explained on the basis of conformational and hydration characteristics of the trehalose molecule. Analyses of the hydration number and radial distribution function of solvent water molecules showed that there was very little hydration adjacent to the glycosidic oxygen of trehalose and that the dynamic conformation of trehalose was less flexible than any of the other sugars due to this anisotropic hydration. The remarkable conformational rigidity that allowed trehalose to act as a sugar template was required for stable interactions with hydrogen-bonded water molecules. Trehalose made an average of 2.8 long-lived hydrogen bonds per each MD step, which was much larger than the average of 2.1 for the other sugars. The stable hydrogen-bond network is derived from the formation of long-lived water bridges at the expense of decreasing the dynamics of the water molecules. Evidence for this dynamic reduction of water by trehalose was also established based on each of the lowest translational diffusion coefficients and the lowest intermolecular coulombic energy of the water molecules around trehalose. Overall results indicate that trehalose functions as a 'dynamic reducer' for solvent water molecules based on its anisotropic hydration and conformational rigidity, suggesting that macroscopic solvent properties could be modulated by changes in the type of glycosidic linkages in sugar molecules.     

Targeted molecular dynamics simulation studies of calcium binding and conformational change in the C-terminal half of gelsolin

Gelsolin consists of six related domains (G1-G6) and the C-terminal half (G4-G6) acts as a calcium sensor during the activation of the whole molecule, a process that involves large domain movements. In this study, we used targeted molecular dynamics simulations to elucidate the conformational transitions of G4-G6 at an atomic level. Domains G4 and G6 are initially ruptured, followed by a rotation of G6 by approximately 90 degrees , which is the dominant conformational change. During this period, local conformational changes occur at the G4 and G5 calcium-binding sites, facilitating large changes in interdomain distances. Alterations in the binding affinities of the calcium ions in these three domains appear to be related to local conformational changes at their binding sites. Analysis of the relative stabilities of the G4-G6-bound calcium ions suggests that they bind first to G6, then to G4, and finally to G5.     

Principal component analysis of DNA oligonucleotide structural data

The microstructure of a DNA helix is characterized by several base pair and base step parameters such as twist, rise, roll, propeller twist, etc., in addition to conformational parameters such as the backbone and the glycosidic torsion angles. Among these only a few, which are independent of all others and of each other, may be used to precisely characterize the helix. The problem however is to identify these independent parameters. We have used principal component analysis to identify a relatively small set of independent parameters, with which to characterize each DNA helix. We show that these principal components clearly discriminate between A and B DNA helical types. The calculations further suggest that the microstructure of a DNA helix is better characterized using dinucleotides.     

Classifier ensembles for protein structural class prediction with varying homology

Structural class characterizes the overall folding type of a protein or its domain. A number of computational methods have been proposed to predict structural class based on primary sequences; however, the accuracy of these methods is strongly affected by sequence homology. This paper proposes, an ensemble classification method and a compact feature-based sequence representation. This method improves prediction accuracy for the four main structural classes compared to competing methods, and provides highly accurate predictions for sequences of widely varying homologies. The experimental evaluation of the proposed method shows superior results across sequences that are characterized by entire homology spectrum, ranging from 25% to 90% homology. The error rates were reduced by over 20% when compared with using individual prediction methods and most commonly used composition vector representation of protein sequences. Comparisons with competing methods on three large benchmark datasets consistently show the superiority of the proposed method.     

A Conformational Change of the γ Subunit Indirectly Regulates the Activity of Cyanobacterial F1-ATPase

The central shaft of the catalytic core of ATP synthase, the γ subunit consists of a coiled-coil structure of N- and C-terminal α-helices, and a globular domain. The γ subunit of cyanobacterial and chloroplast ATP synthase has a unique 30–40-amino acid insertion within the globular domain. We recently prepared the insertion-removed α₃β₃γ complex of cyanobacterial ATP synthase (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855–865). Although the insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition. We postulated that removal of the insertion affects the activity via a conformational change of two central α-helices in γ. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central α-helices to each other by way of a disulfide bond formation. The mutant obtained showed a significant change in ATP hydrolysis activity caused by this restriction. The highly active locked complex was insensitive to N-dimethyldodecylamine-N-oxide, suggesting that the complex is resistant to ADP inhibition. In addition, the lock affected ϵ inhibition. In contrast, the change in activity caused by removal of the γ insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the γ subunit indirectly regulates complex activity by changing both ADP inhibition and ϵ inhibition.     

DNA lesion alters global conformational dynamics of Y-family DNA polymerase during catalysis

A major product of oxidative damage to DNA, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), can lead to genomic mutations if it is bypassed unfaithfully by DNA polymerases in vivo. However, our pre-steady-state kinetic studies show that DNA polymerase IV (Dpo4), a prototype Y-family enzyme from Sulfolobus solfataricus, can bypass 8-oxoG both efficiently and faithfully. For the first time, our stopped-flow FRET studies revealed that a DNA polymerase altered its synchronized global conformational dynamics in response to a DNA lesion. Relative to nucleotide incorporation into undamaged DNA, three of the four domains of Dpo4 undertook different conformational transitions during 8-oxoG bypass and the subsequent extension step. Moreover, the rapid translocation of Dpo4 along DNA induced by nucleotide binding was significantly hindered by the interactions between the embedded 8-oxoG and Dpo4 during the extension step. These results unprecedentedly demonstrate that a Y-family DNA polymerase employs different global conformational dynamics when replicating undamaged and damaged DNA.     

Conformational Selection and Substrate Binding Regulate the Monomer/Dimer Equilibrium of the C-terminal domain of Escherichia coli Enzyme I

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by the binding of phosphoenolpyruvate (PEP) to the C-terminal domain (EIC) of enzyme I (EI), a highly conserved protein that is common to all sugar branches of the PTS. EIC exists in a dynamic monomer/dimer equilibrium that is modulated by ligand binding and is thought to regulate the overall PTS. Isolation of EIC has proven challenging, and conformational dynamics within the EIC domain during the catalytic cycle are still largely unknown. Here, we present a robust protocol for expression and purification of recombinant EIC from Escherichia coli and show that isolated EIC is capable of hydrolyzing PEP. NMR analysis and residual dipolar coupling measurements indicate that the isolated EIC domain in solution adopts a stable tertiary fold and quaternary structure that is consistent with previously reported crystallographic data. NMR relaxation dispersion measurements indicate that residues around the PEP binding site and in the β3α3 turn (residues 333-366), which is located at the dimer interface, undergo a rapid transition on the sub-millisecond time scale (with an exchange rate constant of ～1500 s(-1)) between major open (～97%) and minor closed (～3%) conformations. Upon PEP binding, the β3α3 turn is effectively locked in the closed state by the formation of salt bridges between the phosphate group of PEP and the side chains of Lys(340) and Arg(358), thereby stabilizing the dimer.     

Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.     

Identifying priority areas for reducing species vulnerability to climate change

Aim The dimensions of species vulnerability to climate change are complex, and this impedes efforts to provide clear advice for conservation planning. In this study, we used a formal framework to assess species vulnerability to climate change quantifying exposure, sensitivity and adaptive capacity and then used this information to target areas for reducing vulnerability at a regional scale. Location The 6500‐km² Mount Lofty Ranges region in South Australia. Methods We quantified the vulnerability of 171 plant species in a fragmented yet biologically important agro‐ecological landscape, typical of many temperate zones globally. We specified exposure, using three climate change scenarios; sensitivity, as the adverse impact of climate change on species’ spatial distribution; and adaptive capacity, as the ability of species to migrate calculated using dispersal kernels. Priority areas for reducing vulnerability were then identified by incorporating these various components into a single priority index. Results Climate change had a variable impact on species distributions. Those species whose range decreased or shifted geographically were attributed higher sensitivity than those species that increased geographic range or remained unchanged. The ability to adapt to range changes in response to shifting climates varies both spatially and between species. Areas of highest priority for reducing vulnerability were found at higher altitudes and lower latitudes with increasing severity of climate change. Main conclusions Our study demonstrates the use of a single spatially explicit index that identifies areas in the landscape for targeting specific conservation and restoration actions to reduce species vulnerability to climate change. Our index can be transferred to other regions around the world in which climate change poses an increasing threat to native species.     

How Drugs Interact with Transporters: SGLT1 as a Model

Drugs are transported by cotransporters with widely different turnover rates. We have examined the underlying mechanism using, as a model system, glucose and indican (indoxyl-beta-D: -glucopyranoside) transport by human Na(+)/glucose cotransporter (hSGLT1). Indican is transported by hSGLT1 at 10% of the rate for glucose but with a fivefold higher apparent affinity. We expressed wild-type hSGLT1 and mutant G507C in Xenopus oocytes and used electrical and optical methods to measure the kinetics of glucose (using nonmetabolized glucose analogue alpha-methyl-D: -glucopyranoside, alphaMDG) and indican transport, alone and together. Indican behaved as a competitive inhibitor of alphaMDG transport. To examine protein conformations, we recorded SGLT1 capacitive currents (charge movements) and fluorescence changes in response to step jumps in membrane voltage, in the presence and absence of indican and/or alphaMDG. In the absence of sugar, voltage jumps elicited capacitive SGLT currents that decayed to steady state with time constants (tau) of 3-20 ms. These transient currents were abolished in saturating alphaMDG but only slightly reduced (10%) in saturating indican. SGLT1 G507C rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages. Maximal fluorescence increased approximately 150% in saturating indican but decreased approximately 50% in saturating alphaMDG. Modeling indicated that the rate-limiting step for indican transport is sugar translocation, whereas for alphaMDG it is dissociation of Na(+) from the internal binding sites. The inhibitory effects of indican on alphaMDG transport are due to its higher affinity and a 100-fold lower translocation rate. Our results indicate that competition between substrates and drugs should be taken into consideration when targeting transporters as drug delivery systems.