A General Approach to the Optimization of the Conformation of Ring Molecules with an Application to Valinomycin

Abstract

A general and efficient methodology is presented which allows molecules containing one or many rings of any size to be manipulated within energy minimization procedures. Variables describing the conformation of the molecules concerned are limited to dihedral and ring valence angles and the ring closure conditions are treated as equality constraints. An application is made to the ion transporter valinomycin and its complexes with K⁺ and Na⁺ which illustrates the possibilities of the approach and leads to results which allow a better understanding of the conformational mechanics of this important ionophore.     

Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring

The FtsA protein is a member of the actin superfamily that localizes to the bacterial septal ring during cell division. Deletions of domain 1C or the S12 and S13 beta-strands in domain 2B of the Escherichia coli FtsA, previously postulated to be involved in dimerization, result in partially active proteins that do not allow the normal progression of septation. The truncated FtsA protein lacking domain 1C (FtsADelta1C) localizes in correctly placed division rings, together with FtsZ and ZipA, but does not interact with other FtsA molecules in the yeast two-hybrid assay, and fails to recruit FtsQ and FtsN into the division ring. The rings containing FtsADelta1C are therefore incomplete and do not support division. The production of high levels of FtsADelta1C causes filamentation, an effect that has been reported to result as well from the imbalance between FtsA+ and FtsZ+ molecules. These data indicate that the domain 1C of FtsA participates in the interaction of the protein with other FtsA molecules and with the other proteins that are incorporated at later stages of ring assembly, and is not involved in the interaction with FtsZ and the localization of FtsA to the septal ring. The deletion of the S12-S13 strands of domain 2B generates a protein (FtsADeltaS12-13) that retains the ability to interact with FtsA+. When the mutated protein is expressed at wild-type levels, it localizes into division rings and recruits FtsQ and FtsN, but it fails to sustain septation at normal levels resulting in filamentation. A fivefold overexpression of FtsADeltaS12-13 produces short cells that have normal division rings, but also cells with polar localization of the mutated protein, and cells with rings at abnormal positions that result in the production of a fraction (15%) of small nucleoid-free cells. The S12-S13 strands of domain 2B are not essential for septation, but affect the localization of the division ring.     

Antigen processing for presentation to CD4+ T cells.

The immune system surveys the organism for the presence of foreign or abnormal structures. An important role in the immune response is assumed by T lymphocytes that recognize foreign antigen while tolerating self-proteins. T lymphocytes can recognize only peptide fragments that are presented to them by molecules of the major histocompatibility complex (MHC). Antigen processing for presentation to T cells involves distinct cellular compartments where peptides and MHC molecules interact. Whereas class I MHC molecules (recognized by CD8+ cytotoxic T cells) acquire peptides in an early biosynthetic compartment, class II molecules (recognized by CD4+ helper T cells) acquire peptides most efficiently in an endocytic compartment. It has emerged recently that the class II processing compartment can be fed not only from the outside with exogenous antigen but also from endogenous sources, including membrane-associated and cytosolic proteins. The potential sources of proteins that can trigger a helper T cell response during viral infections and that can induce self-tolerance are thus much wider than previously anticipated.     

Electronic energy transfer and fluorescence quenching in the active sites of mercuric reductase

The FAD-containing enzyme mercuric reductase has been studied by means of steady-state and time-resolved fluorescence spectroscopy. The fluorescence relaxation of the excited state of the isoalloxazine ring of FAD can be described by a sum of two exponential functions. The two lifetimes are not due to a different lifetime of each of the two FAD molecules of mercuric reductase. The FAD molecules are quenched dynamically by a quencher that is not sensitive to the solvent viscosity. In vitro activation induces a dynamic quenching of fluorescence, while upon binding of NADP+ the FAD molecules are both statically and dynamically quenched. Time-resolved fluorescence anisotropy experiments of mercuric reductase in water show that the isoalloxazine ring probably undergoes a rapid and restricted vibrational motion of small amplitude. Electronic energy transfer occurs between the two FAD molecules at a rate of about 3.4 x 10(7) s-1. The angle between the emission transition dipole of the donor and the absorption transition dipole of the acceptor is 137 +/- 2 degrees (or 43 +/- 2 degrees). From previous X-ray data of glutathione reductase we find that the corresponding angle is 160 degrees. This suggests that the isoalloxazine rings of mercuric reductase and glutathione reductase are mutually tilted in slightly different ways.     

Structure-activity relationship of wedelolactone analogues: structural requirements for inhibition of Na+, K+ -ATPase and binding to the central benzodiazepine receptor

Coumestans 2a-i, bearing different patterns of substitution in A- and D-rings, were synthesized and evaluated as inhibitors of kidney Na+, K+ -ATPase and ligands for the central benzodiazepine (BZP) receptor. The presence of a hydroxyl group in position 2 favours the effect on Na+, K+ -ATPase but decreases the affinity for the BZP receptor, allowing the design of more selective molecules than the natural wedelolactone. On the other hand, the presence of a catechol in ring D is important for the effect on both molecular targets.     

Fe- but not Mg-protophorphyrin IX binds to a transmembrane b-type cytochrome

Transmembrane b-type cytochromes, which are crucially involved in electron transfer chains, bind one or more heme (Fe-protoporphyrin IX) molecules non-covalently. Similarly, chlorophylls are typically also non-covalently bound by several membrane integral polypeptides involved in photosynthesis. While both, chlorophyll and heme, are tetrapyrrole macrocycles, they have different substituents at the tetrapyrrole ring moiety. Furthermore, the central metal ion is Mg²⁺ in chlorophyll and Fe^2+/3+ in heme. As heme and chlorophyll a have similar structures and might both be ligated by two histidine residues of a polypeptide chain, and as the local concentration of chlorophyll a might be up to 100-times higher than the concentration of heme, the question arises, as to how an organism ensures specific binding of heme, but not of chlorophyll, to transmembrane apo-cytochromes involved in photosynthetic electron transfer reactions. As shown here, Fe-protoporphyrin IX derivatives with modified substituents at the tetrapyrrole ring moiety still bind to an apo-cytochrome; however, association appears to be reduced. This indicates that hydrophobic and polar interactions of the ring substituents with the protein moiety stabilize the protein/heme-complex but are not essential per se. However, removal or replacement of the central Fe-ion completely abolishes formation of a holo-protein complex, and thus the central iron ion appears to determine heme binding to apo-cytochrome b₆.     

X-ray structure of acarbose bound to amylomaltase from Thermus aquaticus. Implications for the synthesis of large cyclic glucans.

As a member of the alpha-amylase superfamily of enzymes, amylomaltase catalyzes either the transglycosylation from one alpha-1,4 glucan to another or an intramolecular cyclization. The latter reaction is typical for cyclodextrin glucanotransferases. In contrast to these enzymes, amylomaltase catalyzes the formation of cyclic glucans with a degree of polymerization larger than 22. To characterize the factors that determine the size of the synthesized cycloamyloses, we have analyzed the X-ray structure of amylomaltase from Thermus aquaticus in complex with the inhibitor acarbose, a maltotetraose derivative, at 1.9 A resolution. Two acarbose molecules are bound to the enzyme, one in the active site groove at subsite -3 to +1 and a second one approximately 14 A away from the nonreducing end of the acarbose bound to the catalytic site. The inhibitor bound to the catalytic site occupies subsites -3 to +1. Unlike the situation in other enzymes of the alpha-amylase family, the inhibitor is not processed and the inhibitory cyclitol ring of acarbose, which mimicks the half chair conformation of the transition state, does not bind to catalytic subsite -1. The minimum ring size of cycloamyloses produced by this enzyme is proposed to be determined by the distance of the specific substrate binding sites at the active site and near Tyr54 and by the size of the 460s loop. The 250s loop might be involved in binding of the substrate at the reducing end of the scissile bond.     

Binding of adenine and adenine-related compounds to the clay montmorillonite and the mineral hydroxylapatite

The first living things may have consisted of no more than RNA or RNA-like molecules bound to the surfaces of mineral particles. A key aspect of this theory is that these mineral particles have binding sites for RNA and its prebiotic precursors. The object of this study is to explore the binding properties of two of the best studied minerals, montmorillonite and hydroxylapatite, for possible precursors of RNA. The list of compounds investigated includes purines, pyrimidines, nucleosides, nucleotides, nucleotide coenzymes, diaminomaleonitrile and aminoimidazole carbox-amide. Affinities for hydroxylapatite are dominated by ionic interactions between negatively charged small molecules and positively charged sites in the mineral. Binding to montmorillonite presents a more complex picture. These clay particles have a high affinity for organic ring structures which is augmented if they are positively charged. This binding probably takes place on the negatively charged faces of these sheet-like clay particles. Additional binding sites on the edges of of these sheets have a moderate affinity for negatively charged molecules.Small molecules that bind to these minerals sometimes bind independently to sites on the minerals and sometimes bind cooperatively with favorable interactions between the bound molecules.     

Recent studies on the electrospray ionisation mass spectrometric behaviour of selected nitrogen-containing drug molecules and its application to drug analysis using liquid chromatography-electrospray ionisation mass spectrometry

This review presents recent studies on the electrospray ionisation mass spectrometry (ESI-MS) of selected N-containing drug molecules, their metabolites, formulation degradation products and process impurities taken from both studies in the author's laboratory and the recent literature using the Web of Knowledge database. Molecules of mass less than 500 Da are chosen according to selected structural classes in which they give ESI signals primarily in the positive ion mode as [M+H]+ ions. The structural classes are drugs with amine-containing side chains, drugs with N-containing saturated ring structures, drugs with N-containing unsaturated ring structures and quaternary ammonium drugs. Details are given on the fragmentations, where available, that these ionic species exhibit in-source and in ion-trap, triple quadrupole and time-of flight mass spectrometers. Fragmentation data, again where available, using electron impact mass spectrometry (EI-MS) is included for comparison. A review of applications for the period 2004-2005, again taken from the Web of Knowledge database, of the technique liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS) to the detection and determination of these N-containing drug molecules in biomatrices, pharmaceutical formulations, etc., is then made. Analytical information on, for example, sample concentration techniques, LC separation conditions, recoveries from biological media, degradation products and limits of detection (LODs) are provided. Comparisons, where available, are also made with rival analytical techniques such as gas liquid chromatography-mass spectrometry (GLC-MS), capillary electrophoresis-electrospray ionisation mass spectrometry (CE-ESI-MS) and stripping voltammetry (SV).     

Spatial resolution of two bacterial cell division proteins: ZapA recruits ZapB to the inner face of the Z‐ring

FtsZ, the essential regulator of bacterial cell division, is a dynamic cytoskeletal protein that forms helices that condense into the Z‐ring prior to division. Two small coiled‐coil proteins, ZapA and ZapB, are both recruited early to the Z‐ring. We show here that ZapB is recruited to the Z‐ring by ZapA. A direct interaction between ZapA and ZapB is supported by bacterial two‐hybrid and in vitro interaction assays. Using high‐resolution 3‐D reconstruction microscopy, we find that, surprisingly, ZapB is located inside the Z‐ring in virtually all cells investigated. We propose a molecular model in which ZapA increases lateral interactions between FtsZ proto‐filaments and ZapB mediates further stabilization of this interaction by cross‐linking ZapA molecules bound to adjacent FtsZ proto‐filaments. Gene deletion and complementation assays show that ZapB can mitigate cell division and Z‐ring assembly defects even in the absence of ZapA, raising the possibility that ZapB stimulates Z‐ring assembly by two different mechanisms.     

Kinetics of coenzyme binding to liver alcohol dehydrogenase in the pH range 10-12

On- and off-velocity constants for NADH and NAD+ binding to liver alcohol dehydrogenase in the pH range 10-12 have been determined by stopped-flow kinetic methods. The results are consistent with previously reported equilibrium binding data and proposals attributing the main effects of pH on coenzyme binding to ionization of Lys-228 and zinc-bound water. Deprotonation of the group identified as Lys-228 decreases the NADH and NAD+ association rates by a factor exceeding 20 and has no detectable effect on the coenzyme dissociation rates in the examined pH range. Ionization of the group identified as zinc-bound water causes a 3-fold increase of the rate of NADH dissociation from the enzyme, and decreases the rate of NAD+ dissociation by a factor of 200. The NADH and NAD+ association rates are decreased by a factor of 30 and 5, respectively. The observed effects of pH can be rationalized in terms of electrostatic interactions of the ionizing groups with the charges present on the coenzyme molecules and lend support to the idea that binding of the coenzyme nicotinamide ring occurs subsequent to binding of the AMP portion of the coenzyme.     

Cytokinesis: welcome to the Rho zone

Cytokinesis follows nuclear division and generates two distinct daughter cells, each replete with a full complement of the genome and cytoplasmic organelles. Members of the Rho family of GTPases are crucial regulators of this process in a wide variety of species. In many cell types, cytokinesis is mediated by a discretely localized contractile ring that is rich in actin and myosin. In this article (which is part of the Cytokinesis series), we review recent studies in animal cells that have shown that local assembly of the contractile ring is mediated by a discrete pool of GTP-bound, active RhoA. Advances in detecting the active pool of RhoA have allowed insights into the mechanisms and the molecules that promote the accumulation of active RhoA at the correct time and place in the cell.     

Nuclear Overhauser effect studies of the conformation of Co(NH3)4ATP bound to kidney Na,K-ATPase

Transferred nuclear Overhauser effect measurements (in the two-dimensional mode) have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase [Klevickis, C., & Grisham, C.M. (1982) Biochemistry 21, 6979. Gantzer, M.L., et al. (1982) Biochemistry 21, 4083]. Nine unique proton-proton distances on ATPase-bound Co(NH3)4ATP were determined from the initial build-up rates of the cross-peaks of the 2D-TRNOE data sets. These distances, taken together with previous 31P and 1H relaxation measurements with paramagnetic probes, are consistent with a single nucleotide conformation at the active site. The bound Co(NH3)4ATP) adopts an anti conformation, with a glycosidic torsion angle of 35 degrees, and the conformation of the ribose ring is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this conformation are 100 degrees and 178 degrees, respectively. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. Mn2+ bound to a single, high-affinity site on the ATPase lies above and in the plane of the adenine ring. The distances from enzyme-bound Mn2+ to N6 and N7 are too large for first coordination sphere complexes, but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium binding cyclic hexapeptide. Crystal structure of cyclo-(L-prolyl-glycyl)3 calcium complex

The synthetic cyclic hexapeptide (L-prolyl-glycyl)3 forms a 2:1 complex with Ca2+ ion. The cation is sandwiched between the two peptide molecules. The glycyl carbonyls from each of the peptides are octahedrally coordinated to the cation with an average calcium oxygen coordination distance of 2.26A. Both the molecules coordinating to the calcium ion have three fold symmetry, but show significant conformational differences. In one of the peptides of the sandwich, the alternate carbonyls point to the opposite sides of the peptide ring while in the other, all the six carbonyls point to the same side of the ring. Three NH ... O hydrogen bonds between the peptides add to the stability of the sandwich.     

A new analysis of the depolymerized fragments of lignin polymer using ToF-SIMS

Lignin in plant cell walls is a complex, irregular polymer built from phenylpropanoid C6-C3 units that are connected via various C-C and C-O linkages. A recent study using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with Ga primary ion bombardment showed that lignin polymers can be characterized by specific positive ions possessing a substituted aromatic ring (so-called guaiacyl or syringyl rings), which are the basic building units of lignin. To study the relationship between the characteristic ions of lignin and the common interunit linkages, various lignin dimer model compounds were investigated using ToF-SIMS. The resulting dimer spectra showed that the characteristic ions with a guaiacyl ring at m/z 137 and 151 result from rupture of most common interunit linkages, not only 8-O-4' linkages, which are the most abundant in lignin, but also 8-1', 8-5', and 8-8'. There was no evidence of rupture of 5-5' linkages. These results show that ToF-SIMS offers a new tool for the direct analysis of the depolymerized fragments of lignin polymers. The mechanisms for the fragmentation of lignin dimer models in ToF-SIMS were proposed that allow ToF-SIMS fragmentation rules to be deduced. Adduct ions such as [M + 13]+ ([M + CH]+) were also produced in fragmentation of the dimers and are thought to arise from the combination of the molecules with their stable fragments.     

Monte Carlo Simulations of the Chemical Potential and Free Energy for Trimer and Hexamer Rings

Abstract

The chemical potential of a trimer and hexamer model ring system was determined by computer simulation over a range of temperatures and densities. Such ring molecules are important as model aromatic and naphthenic hydrocarbons. Thermodynamic integration of the pressure along a reversible path, Widom's ghost particle insertion method and Kirkwood's charging parameter method were used over a molecular density range of 0.05 to 0.30. Data were obtained by Monte Carlo simulation of a 96 molecule system that was modelled with a Lennard-Jones 6-12 truncated potential. The original insertion method, which does not take into account the orientation of the molecule when it is inserted, gives results for the chemical potential which deviate from that obtained using the thermodynamic pressure integration. At high density or temperature the deviation is significant. We have modified the Widom insertion technique to account for this short range orientation and find good agreement between this technique and the thermodynamic integration method for the chemical potential. We also calculated the free energy difference between our model ring molecules and ring molecules made up of hard spheres.     

A nuclear magnetic resonance study of the metal binding sites in bacitracin.

Carbon-13 nuclear magnetic resonance spectroscopy has been used to identify sites in bacitracin which bind Cu2+ and Mn2+. Results are presented which implicate the free carboxyl groups of the aspartic and glutamic acid residues and the imidazole ring of the histidine residue as metal complexation sites between pH 6 and 8. Evidence is presented which also indicates that the thiazoline ring of bacitracin binds Mn2+. Bacitracin does not bind Cu2+ or Mn2+ at pH values of 2.5 or less.     

The X-ray structure and absolute configuration of the anticancer drug (+)-4-ketocyclophosphamide

4-Ketocyclophosphamide (4-keto CP), C7H12Cl2N2O3P, monoclinic, P2(1), a = 11.909 (2), b = 10.254 (1), c = 9.873 (1) A, beta = 91.08 (1) degrees, V = 1205.45 (3) A3 Z = 4, Dc = 1.51 Mg-m-3, Cu K alpha, lambda = 1.54178 A, alpha 25 = +53.8 degrees (c = 3.0, MeOH), m.p. 107 degrees C, mu = 61.8 cm-1, F (000) = 564, R = 0.064 for 2961 observed reflexions with I greater than 1.96 sigma(I). Dextrarotatory enantiomer of 4-keto CP has S configuration at the stereogenic center. One of the two crystallographically independent molecules is disordered both in a six-membered ring and in --N(CH2CH2Cl)2 moiety. With the exception of a less populated conformer of a disordered molecule, 4-keto CP molecules adopt a conformation in which 1,3,2-oxazophosphorinane ring is in the sofa form with C(6) deviating from the plane through the remaining five ring atoms while an exocyclic N atom with its three substituents is nearly coplanar with the phosphoryl oxygen atom O(8). In a less populated conformer, the six membered ring takes the form of sofa with C(5) as a flap while an exocyclic N atom and its substituents are oriented toward the P--N(3) bond.     

Function of two beta-carotenes near the D1 and D2 proteins in photosystem II dimers

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 A resolution), in which 11 beta-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (Em) of one-electron oxidation for all Car (Em(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (CarD1) in van der Waals contact with the chlorin ring of ChlZ(D1). Each CarD1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between CarD1 and plastoquinone/Q(A) may imply a direct charge recombination of Car+Q(A)-. The calculated Em(CarD1) and Em(ChlZ(D1)) are, respectively, 83 and 126 mV higher than Em(CarD2) and Em(ChlZ(D2)), which could explain why CarD2+ and ChlZ(D2)+ are observed rather than the corresponding CarD1+ and ChlZ(D1)+.     

From hopanoids to cholesterol: Molecular clocks of pentameric ligand-gated ion channels

Pentameric ligand-gated ion channels (pLGICs) and their lipid microenvironments appear to have acquired mutually adaptive traits along evolution: 1) the three-ring architecture of their transmembrane (TM) region; 2) the ability of the outermost TM ring to convey lipid signals to the middle ring, which passes them on to the central pore ring, and 3) consensus motifs for sterol recognition in all pLGICs. Hopanoids are triterpenoid fossil lipids that constitute invaluable biomarkers for tracing evolution at the molecular scale. The cyanobacterium Gloeobacter violaceus is the oldest known living organism in which the X-ray structure of its pLGIC, GLIC, reveals the presence of the above attributes and, as discussed in this review, the ability to bind hopanoids. ELIC, the pLGIC from the bacillus Erwinia chrysanthemi is the only other known case to date. Both prokaryotes lack cholesterol but their pLGICs exhibit the same sterol motifs as mammalian pLGIC. This remarkable conservation suggests that the association of sterols and hopanoid surrogate molecules arose from the early need in prokaryotes to stabilize pLGIC TM regions by means of relatively rigid lipid molecules. The conservation of these phenotypic traits along such a long phylogenetic span leads us to suggest the possible co-evolution of these sterols with pLGICs.