Parallel Double Helices of DNA. Conformational Analysis of Regular Helices with the Second Order Symmetry Axis

Abstract

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands connected with the second order symmetry axis being at the same time the helix axis. The calculations were made for homopolymers poly(dA) · poly(dA), poly(dC) · poly(dC), poly(dG) poly(dG), and poly(dT) · poly(dT). All possible variants of hydrogen bonding of base pairs of the same name were studied for each polymer. The maps of backbone chain geometrical existence were constructed. Conformational and helical parameters corresponding to local minima of conformational energy of “parallel” DNA helices, calculated at atom-atom approximation, were determined. The dependence of conformational energy on the base pair and on the hydrogen bond type was analysed. Two major conformational advantageous for “parallel” DNA's do not depend much on the hydrogen-bonded base pair type were indicated. One of them coincided with the conformational region typical for “antiparallel” DNA in particular for the B-form DNA Conformational energy of “parallel” DNA depends on the base pair type and for the most part is similar to the conformational energy of “antiparallel” B-DNA.     

Conformational Changes in Talin on Binding to Anionic Phospholipid Membranes Facilitate Signaling by Integrin Transmembrane Helices

Integrins are heterodimeric (αβ) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/β integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/β TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and β transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation.     

Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca²⁺ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca²⁺ binding domain, we also studied the occlusion of Ca²⁺, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca²⁺ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E₁-E₂ model devised using kinetics methodology only.     

Calcium Ion Binding Properties of Medicago truncatula Calcium/Calmodulin-Dependent Protein Kinase

A calcium/calmodulin-dependent protein kinase (CCaMK) is essential in the interpretation of calcium oscillations in plant root cells for the establishment of symbiotic relationships with rhizobia and mycorrhizal fungi. Some of its properties have been studied in detail, but its calcium ion binding properties and subsequent conformational change have not. A biophysical approach was taken with constructs comprising either the visinin-like domain of Medicago truncatula CCaMK, which contains EF-hand motifs, or this domain together with the autoinhibitory domain. The visinin-like domain binds three calcium ions, leading to a conformational change involving the exposure of hydrophobic surfaces and a change in tertiary but not net secondary or quaternary structure. The affinity for calcium ions of visinin-like domain EF-hands 1 and 2 (K(d) = 200 ± 50 nM) was appropriate for the interpretation of calcium oscillations (～125-850 nM), while that of EF-hand 3 (K(d) ≤ 20 nM) implied occupancy at basal calcium ion levels. Calcium dissociation rate constants were determined for the visinin-like domain of CCaMK, M. truncatula calmodulin 1, and the complex between these two proteins (the slowest of which was 0.123 ± 0.002 s(-1)), suggesting the corresponding calcium association rate constants were at or near the diffusion-limited rate. In addition, the dissociation of calmodulin from the protein complex was shown to be on the same time scale as the dissociation of calcium ions. These observations suggest that the formation and dissociation of the complex between calmodulin and CCaMK would substantially mirror calcium oscillations, which typically have a 90 s periodicity.     

Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs

An important step in the production of inflammatory mediators of the leukotriene family is the Ca²⁺ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca²⁺ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron microscopy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.     

Metal Ion Binding and Conformational Transitions in Concanavalin A: A Structure-Function Study

Abstract

The affinity of the lectin Concanavalin A (Con A) for saccharides, and its requirement for metal ions such as Mn²⁺ and Ca²⁺, have been known for about 50 years. However the relationship between metal ion binding and the saccharide binding activity of Con A has only recently been examined in detail. Brown et al. (Biochemistry 16, 3883 (1977)) showed that Con A exists as a mixture of two conformational states: a “locked” form and an “unlocked” form. The unlocked form of the protein weakly binds metal ions and saccharide, and is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. Brown and coworkers measured the kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn²⁺and Ca²⁺. They also demonstrated that Mn²⁺ alone could form a locked ternary complex with Con A, and that rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25°C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin.

Based on the kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data, it appears that the transition between the two conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near one of the two metal ion binding sites. The relatively large activation energy for the transition (～ 22 kcal M^?1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the two conformations studied.

We have identified and characterized binary, ternary, and quaternary complexes of both conformations of Con A containing Mn²⁺ and saccharide, and have determined both metalion and saccharide dissociation constants for all of them, as well as equilibrium and kinetic values for the conformational transitions between them. The main finding is that saccharide binds very weakly (K_d～2 M) to unlocked apo-Con A and very tightly to the locked ternary Mn²⁺-Con A complex (K_d～ 10^?4 M). Saccharide binding increases along the various pathways connecting these two species in a nonadditive fashion. Thus, both conformation and metal ion binding determine the saccharide affinity of each complex, although the specificity of saccharide binding of the various species is maintained throughout.     

Effects of Calcium Ion Concentration on Cel Wall Synthesis

Cuttings of 6-week-old Norway spruce (Picea abies (L.) Karst.)seedlings were placed in liquid media containing various concentrationsof Ca²⁺. Cytoplasmic concentrations of Ca²⁺ were manipulatedusing the ionophore A 23 187. The effects of Ca²⁺ concentrationson the deposition of total cell wall material as well as onthe deposition of cellulose, lignin, and non-cellulosic polysaccharidesin the hypocotyls were investigated. At low concentrations ofCa²⁺ wall deposition was reduced, mainly as a result of theinhibition of lignin and non-cellulosic polysaccharide deposition.High concentrations of Ca²⁺ stimulated non-cellulosic polysaccharideand lignin deposition, whereas cellulose deposition was almosttotally inhibited. Key words: Conifers, calcium, cell wall, lignin, cellulose     

Minimalistic Predictor of Protein Binding Energy: Contribution of Solvation Factor to Protein Binding

It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software.     

Effects of Calcium Ion on Growth and Behavior of Tetrahymena pyriformis

The chelator GEDTA was used to show that Ca²⁺ is required for the growth of Tetrahymena pyriformis strain W and for normal galvanotactic responses and swimming.     

Ion Effects on Calcium Accumulation by Cardiac Sarcoplasmic Reticulum

The effects of monovalent cations on the active calcium-accumulating ability of cardiac sarcoplasmic reticulum were assessed. Grana prepared in an ion-free system accumulated calcium when ATP and Mg⁺⁺ were present. Sodium ion and to a lesser extent lithium but not K⁺ reduced the amount of calcium taken up. The reduction of calcium binding by Na⁺ is not due to inhibition of uptake but to a rapid release of the radiocalcium bound. The amount of calcium released by sodium does not appear to be enough to explain contraction on the basis of sodium influx into muscle, but may be significant in the regulation of tension.     

The Influence of Hydrogen Ion Concentration on Calcium Binding and Release by Skeletal Muscle Sarcoplasmic Reticulum

Calcium release and binding produced by alterations in pH were investigated in isolated sarcoplasmic reticulum (SR) from skeletal muscle. When the pH was abruptly increased from 6.46 to 7.82, after calcium loading for 30 sec, 80–90 nanomoles (nmole) of calcium/mg protein were released. When the pH was abruptly decreased from 7.56 to 6.46, after calcium loading for 30 sec, 25–30 nmole of calcium/mg protein were rebound. The calcium release process was shown to be a function of pH change: 57 nmole of calcium were released per 1 pH unit change per mg protein. The amount of adenosine triphosphate (ATP) bound to the SR was not altered by the pH changes. The release phenomenon was not due to alteration of ATP concentration by the increased pH. Native actomyosin was combined with SR in order to study the effectiveness of calcium release from the SR by pH change in inducing super-precipitation of actomyosin. It was found that SR, in an amount high enough to inhibit superprecipitation at pH 6.5, did not prevent the process when the pH was suddenly increased to 7.3, indicating that the affinity of SR for calcium depends specifically on pH. These data suggest the possible participation of hydrogen ion concentration in excitation-contraction coupling.     

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: I. Sodium Binding is Associated with a Conformational Rearrangement

To investigate Na⁺ binding to the ion-binding sites presented on the cytoplasmic side of the Na,K-ATPase, equilibrium Na⁺-titration experiments were performed using two fluorescent dyes, RH421 and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na⁺ in the presence of various Mg²⁺ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na⁺ ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na⁺ ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na⁺ ions and followed by the conformational transition E₁/E₂ of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na⁺ ions are bound. Received: 8 October 1998/Revised: 29 December 1998     

Effects of Calcium on the Binding of Phencyclidine to Acetylcholine Receptor-Rich Membrane Fragments from Torpedo californica Electroplaque

Abstract: The influence of calcium on the binding of phencyclidine (PCP) to acetylcholine (ACh) receptor-rich membrane fragments was investigated. Calcium decreased the equilibrium affinity for PCP in the presence, but not in the absence, of the cholinergic agonist carbamylcholine. The effect of calcium was rapidly reversible by EGTA, indicating that it was not attributable to a calcium-activated protease or a phospholipase. Following detergent solubilization of the nicotinic ACh receptor, the calcium effect on PCP remained, suggesting that calcium may interact directly with the receptor to exert its effect. Other divalent cations (Mn²⁺, La²⁺ Co²⁺, Mg²⁺) had similar effects. A correlate of "desensitization" of the ACh receptor can be observed using PCP binding, and a two-step "desensitization" process can be observed. Calcium seemed to increase the amplitude of a rapid component of receptor "desensitization." The results presented in this paper suggest that calcium may play a role in the modulation of the nicotinic ACh receptor.     

Effects of Calcium Ion and the Protective Action on Survival and Growth Inhihition of Pollen

Studies were conducted on the protective action of Ca against growth inhibition and death of pollen from Crinum asiaticum and Catharanthus roseus. Ca ions not only promoted pollen germination and pollen tube growth, but also were antagonistic to pollen injuries induced by various chemical and physical treatments. Most of the chemicals used seemed to act as osmotic inhibitors. Among the chemical agents tested, DNP inhibited pollen germination more strikingly with Ca than without it, whereas pollen tube growth was enhanced. Another metabolic inhibitor, low temperature, showed a similar effect on pollen germination. Insignificant or no protective action of Ca was observed when IAA, TIBA and coumarin, were applied. Large populations of pollen when grown in cultural media witbout Ca showed a partial protective action. The protective action of Ca in pollen growth against various inhibitors required other cations such as Mg and K ions. The promoting of pollen growth by Ca also required tbese otber cations. The protective action of Ca is considered to be based on its binding in tbe pollen cell walls, particularly in the pectic regions. This gives rise to a decreased permeability and increased structural rigidity against tbe chemical and physical inhibitors.     

Stabilization of DNA Double and Triple Helices by Conjugation of Minor Groove Binders to Oligonucleotides

Abstract

New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.     

Ion and Temperature Effects on the Binding of γ-Aminobutyrate to Its Receptors and the High-Affinity Transport System

A set of procedures was developed to study the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to GABAA and GABAB receptors, and to the Na(+)-dependent transport carrier, at 25 and 37 degrees C in the presence of physiological concentrations of Na+. The membrane preparation used in these procedures was not subjected to freeze-thawing or treatment with Triton X-100. Isoguvacine, (-)-baclofen, and (-)-nipecotate were used to block selectively the binding to GABAA receptors, GABAB receptors, and the transport site, respectively. Analysis of the binding characteristics of [3H]GABA to the GABAA receptor suggested the existence of high-(KD less than 30 nM), middle- (KD = 100-500 nM), and low-affinity (KD greater than 5 microM) binding sites. However, the binding data in the middle-affinity region (100-1,000 nM) were often indicative of cooperativity. The affinity between GABA and the GABAA receptor was reduced modestly by increases in temperature and by the presence of Cl- at physiological concentrations. Binding to the GABAB receptor required Ca2+ and Cl-. Apparent binding to the transport carrier required both Na+ and Cl-. A comparison of Bmax values in three brain regions revealed an inverse relationship between the high-affinity site of the GABAA receptor and the transport binding site.     

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca²⁺) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca²⁺ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca²⁺ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca²⁺ affinity to a membrane-associated, higher Ca²⁺ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca²⁺ influx is the basis for the cooperative response of annexin a5 toward Ca²⁺, and the role of membrane organization in this response.     

Variability in the Geometry of RNA Double Helices Generated from Dinucleoside Building-Blocks

Abstract

A graphical method is presented for the generation of helical parameters from single-crystal structures of RNA nucleic acid fragments that are minimally dinucleosides. The method is compared with other published procedures, for a number of text examples. The RNA double helices generated from three different salts of the dinucleoside monophosphate GpC are examined in relation to the variations in helix morphology that are produced. It is shown that small differences between these GpC salts can be amplified to very distinct helix characteristics.     

Conformational Studies on Calcium Binding By tBoc-Leu-Pro-Tyr-Ala-NHCH3, a Tyrosine Kinase Substrate,in a Nonpolar Solvent

Abstract

With a view to understanding the structural requirement for tyrosine phosphorylation, we have examined the free and Ca²⁺-bound conformations of the synthetic peptide tBoc-Leu-Pro-Tyr-Ala-NHCH₃, a substrate for a protein tyrosine kinase, using circular dichroism (CD), ¹H and ¹³C nuclear magnetic resonance (NMR) and molecular modeling methods. CD spectrum of the free peptide in water showed a random coil structure, while the spectrum in acetonitrile was indicative of a folded structure containing a type III β-turn. Dihedral angle data derived from J_NH-CH coupling constants, as well as two-dimensional ¹H-COSY and NOESY spectral analyses, showed that the peptide adopts a conformation close to the 3_10- helix. Ca²⁺ binding by the peptide, as monitored by CD spectral changes, was quite weak in water. However, substantial CD spectral changes were observed in the peptide on addition of Ca²⁺ in acetonitrile suggestive of major conformational alterations due to Ca²⁺ binding. Analysis of the binding isotherms at 25°C obtained from CD data in acetonitrile indicated a 2:1 peptide:Ca²⁺ (“sandwich”) complex to be the dominant species with a K_d of about 30μM. A. 1:1 complex was also present and became significant at Ca²⁺:peptide ratios above 1. By comparison, the peptide formed a predominantly 1:1 complex with Mg²⁺ with a K_d of about 40μM. ¹³C-NMR data showed that a mixture of cis and trans conformers (arising from rotation around the Leu-Pro bond) in the free peptide changes over to the all-trans form on coordination of the peptide carbonyl groups to the Ca²⁺ ion. ¹H-NOESY data of the Ca²⁺ complex revealed several interactions involving the sidechains of two peptide molecules in the sandwich. Molecular modeling and energy minimization with and without the input of NOESY-derived distance constraints showed the sandwich complex to be an energetically very favourable conformation. Besides its relevance in terms of the possible involvement of divalent cations in substrate-tyrosine kinase interaction, the conformational characterization of tBoc-Leu-Pro-Tyr-Ala-NHCH₃ and its Ca²⁺ complex should help understand the conformational determinants for Ca²⁺-binding by linear peptides.