-
[show abstract]
[hide abstract]
ABSTRACT: The biological information encoded in carbohydrate sequences dwarfs that of proteins and nucleic acids. Deciphering structure-function relationships in heparin and heparan sulfate (the heparanome) is further compounded by extreme sequence diversity, experimental difficulties and the computational cost of rigorous modeling. Here, we perform unbiased microsecond dynamics simulations of 11 heparanome oligosaccharides (55 microseconds total) to investigate the effect of sequence on 3D-structure and to underpin a coarse-grained model that is consistent with long-timescale experimentally-validated atomic motions in water. Pyranose ring flexing (puckering) in 2-O-sulfo-α-L-iduronic acid, which underlies heparin-mediated anticoagulation, was modulated by polymerization (chain position and adjacent residues), which is supported by previous experiments. Furthermore, in coarse-grained simulations inclusion of puckering was essential to predict macroscopic hydrodynamic properties of heparan sulfate chains containing hundreds of monosaccharaides. Our structural findings and model enable rational molecular design and we propose that in the heparanome, puckering, polymer 3D-shape and bioactivity are inextricably linked.
Biomacromolecules 02/2013; · 5.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Reconciling glycocodes and their associated bioactivities, via 3D-structure, will rationalise burgeoning high-throughput functional glycomics data and underpin a new era of opportunity in chemical biology. A major impasse to achieving this goal is a detailed understanding of pyranose sugar ring 3D-conformation (or pucker) and the affiliated microsecond-timescale exchange kinetics. Here, we perform hardware-accelerated kinetically-rigorous equilibrium simulations of fundamental monosaccharides to produce the hypothesis that pyranoses have microsecond-timescale kinetic puckering signatures in water, classified as unstable (rare in the glycome), metastable (infrequently observed) and stable (prevalent). The predicted μs-metastability of β-d-glucose explained hitherto irreconcilable experimental measurements. Twisted puckers seen in carbohydrate enzymes were present in the aqueous 3D-ensemble (suggesting preorganization) and pyranose-water interactions accounted for the relative stability of β-d-galactose. Characteristic 3D-shapes for biologically- and commercially-important carbohydrates and new rules linking chemical modifications with pyranose μs-puckering kinetics are proposed. The observations advance structural-glycomics towards dynamic 3D-templates suitable for structure-based design.
Physical Chemistry Chemical Physics 03/2012; 14(16):5843-8. · 3.57 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Understanding microsecond-timescale dynamics is crucial to establish three-dimensional (3D) structure-activity relationships in sugars but has been intractable to experiments and simulations. As a consequence, whether arguably the most important chemical scaffold in glycobiology, N-acetyl-d-glucosamine (GlcNAc), deviates from a rigid (4)C(1) chair is unknown. Here, conformer populations and exchange kinetics were quantified from the longest aqueous carbohydrate simulations to date (0.2 ms total) of GlcNAc, four derivatives from heparan sulfate and their methylglycosides. Unmodified GlcNAc took 3-5 μs to reach a conformational equilibrium, which comprised a metastable (4)C(1) chair that underwent (4)C(1) ↔ (1)C(4) transitions at a predicted forward rate of 0.8 μs(-1) with an average (1)C(4)-chair lifetime of 3 ns. These predictions agree with high-resolution crystallography and nuclear magnetic resonance but not with the hypothesis that GlcNAc is a rigid (4)C(1) chair, concluded from previous experimental analyses and non-aqueous modeling. The methylglycoside was calculated to have a slower forward rate (0.3 μs(-1)) and a more stable (4)C(1) conformer (0.2 kcal mol(-1)), suggesting that pivotal 3D intermediates (particularly (2)S(O), (1)S(5) and B(2,5)) increased in energy, and water was implicated as a major cause. Sulfonation (N-, 3-O and 6-O) significantly augmented this effect by blocking pseudorotation, but did not alter the rotational preferences of hydroyxl or hydroxymethyl groups. We therefore propose that GlcNAc undergoes puckering exchange that is dependent on polymerization and sulfo substituents. Our analyses, and 3D model of the equilibrium GlcNAc conformer in water, can be used as dictionary data and present new opportunities to rationally modify puckering and carbohydrate bioactivity, with diverse applications from improving crop yields to disease amelioration.
Glycobiology 08/2011; 21(12):1651-62. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The 3D-structure of extracellular matrix glycosaminoglycans is central to function, but is currently poorly understood. Resolving this will provide insight into their heterogeneous biological roles and help to realize their significant therapeutic potential. Glycosaminoglycan chemical isoforms are too numerous to study experimentally and simulation provides a tractable alternative. However, best practice for accurate calculation of glycosaminoglycan 3D-structure within biologically relevant nanosecond timescales is uncertain. Here, we evaluate the ability of three potentials to reproduce experimentally observed glycosaminoglycan monosaccharide puckering, disaccharide 3D-conformation, and characteristic solvent interactions. Temporal dynamics of unsulfated chondroitin, chondroitin-4-sulfate, and hyaluronan β(1→3) disaccharides were simulated within TIP3P explicit solvent unrestrained for 20 ns using the GLYCAM06 force-field and two semi-empirical quantum mechanics methods, PM3-CARB1 and SCC-DFTB-D (both within a hybrid QM/MM formalism). Comparison of calculated and experimental properties (vicinal couplings, nuclear Overhauser enhancements, and glycosidic linkage geometries) showed that the carbohydrate-specific parameterization of PM3-CARB1 imparted quantifiable benefits on monosaccharide puckering and that the SCC-DFTB-D method (including an empirical correction for dispersion) best modeled the effects of hexosamine 4-sulfation. However, paradoxically, the most approximate approach (GLYCAM06/TIP3P) was the best at predicting monosaccharide puckering, 3D-conformation, and solvent interactions. Our data contribute to the debate and emerging consensus on the relative performance of these levels of theory for biological molecules.
Journal of Computational Chemistry 12/2010; 31(16):2932-47. · 4.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The pyranose ring of L-iduronic acid (IdoA), a major constituent of the anticoagulant heparin, is an equilibrium of multiple ring puckers that have evaded quantification by experiment or computation. In order to resolve this enigma, we have calculated the free energy landscape of IdoA and two related monosaccharides from extensive microsecond simulations. After establishing that the simulated puckers had reached equilibrium, hypotheses were confirmed that (a) IdoA (1)C(4)- and (4)C(1)-chair conformations exchange on the microsecond time scale, (b) C5 epimerization leads to a (4)C(1)-chair, and (c) IdoA 2-O-sulfation (IdoA2S) stabilizes the (1)C(4) conformer. The IdoA and IdoA2S (1)C(4) conformers were isoenergetic and computed to be 0.9 and 2.6 kcal mol(-1) lower in free energy than their respective (4)C(1)-chair conformations. The simulations also predicted that the IdoA (2)S(O)-skew-boat was less populated than previously thought. Novel chemical synthesis and ultra-high-field NMR supported these observations, but slight discrepancies in observed and predicted NMR vicinal couplings implied that the simulation overestimated the population of the IdoA (4)C(1)-chair with respect to (1)C(4)-chair due to small force field inaccuracies that only manifest in long simulations. These free-energy calculations drive improvements in computational methods and provide a novel route to carbohydrate mimetic biomaterials and pharmaceuticals.
Journal of the American Chemical Society 09/2010; 132(38):13132-4. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-structure at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without (15)N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the beta(1-->3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the beta(1-->3) psi dihedral (as previously predicted by simulation), while the beta(1-->4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties.
Carbohydrate research 11/2009; 345(2):291-302. · 2.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The human alpha7 nicotinic acetylcholine receptor (nAChR) subunit and its Caenorhabditis elegans homolog, ACR-16, can generate functional recombinant homomeric receptors when expressed in Xenopus laevis oocytes. Both nAChRs express robustly in the presence of the co-injected chaperone, RIC-3, and show striking differences in the actions of a type I positive allosteric modulator (PAM), ivermectin (IVM). Type I PAMs are characterised by an increase in amplitude only of the response to acetylcholine (ACh), whereas type II PAMs exhibit, in addition, changes in time-course/desensitization of the ACh response. The type I PAMs, ivermectin, 5-hydroxyindole (5-HI), NS-1738 and genistein and the type II PAM, PNU-120596, are all active on human alpha7 but are without PAM activity on ACR-16, where they attenuate the amplitude of the ACh response. We used the published structure of avermectin B1a to generate a model of IVM, which was then docked into the candidate transmembrane allosteric binding site on alpha7 and ACR-16 in an attempt to gain insights into the observed differences in IVM actions. The new pharmacological findings and computational approaches being developed may inform the design of novel PAM drugs targeting major neurological disorders.
Biochemical pharmacology 07/2009; 78(7):836-43. · 4.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The molecular diversity of many gene products functioning in the nervous system is enhanced by alternative splicing and adenosine-to-inosine editing of pre-mRNA. Using RDL, a Drosophila melanogaster GABA-gated ion channel, we examined the functional impact of RNA editing at several sites along with alternative splicing of more than one exon. We show that alternative splicing and RNA editing have a combined influence on the potency of the neurotransmitter GABA, and the editing isoforms detected in vivo span the entire functional range of potencies seen for all possible edit variants expressed in Xenopus laevis oocytes. The extent of RNA editing is developmentally regulated and can also be linked to the choice of alternative exons. These results provide insights into how the rich diversity of signaling necessary for complex brain function can be achieved by relatively few genes.
Journal of Neuroscience 05/2009; 29(13):4287-92. · 7.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Many of the insecticides in current use act on molecular targets in the insect nervous system. Recently, our understanding of these targets has improved as a result of the complete sequencing of an insect genome, i.e., Drosophila melanogaster. Here we examine the recent work, drawing on genetics, genomics and physiology, which has provided evidence that specific receptors and ion channels are targeted by distinct chemical classes of insect control agents. The examples discussed include, sodium channels (pyrethroids, p,p'-dichlorodiphenyl-trichloroethane (DDT), dihydropyrazoles and oxadiazines); nicotinic acetylcholine receptors (cartap, spinosad, imidacloprid and related nitromethylenes/nitroguanidines); gamma-aminobutyric acid (GABA) receptors (cyclodienes, gamma-BHC and fipronil) and L-glutamate receptors (avermectins). Finally, we have examined the molecular basis of resistance to these molecules, which in some cases involves mutations in the molecular target, and we also consider the future impact of molecular genetic technologies in our understanding of the actions of neuroactive insecticides.
Invertebrate Neuroscience 12/2005; 5(3-4):119-33. · 1.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The reactions of several active site mutant forms of bacterial morphinone reductase (MR) with NADH and 2-cyclohexen-1-one as substrates have been studied by stopped-flow and steady-state kinetic methods and redox potentiometry. The enzymes were designed to (i) probe a role for potential proton donors (Tyr-72 and Tyr-356) in the oxidative half-reaction of MR; (ii) assess the function of a highly conserved tryptophan residue (Trp-106) in catalysis; (iii) investigate the role of Thr-32 in modulating the FMN reduction potential and catalysis. The Y72F and Y356F enzymes retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key proton donors in the oxidative reaction of MR. Taken together with our recently published data (Messiha, H. L., Munro, A. W., Bruce, N. C., Barsukov, I., and Scrutton, N. S. (2005) J. Biol. Chem. 280, 4627-4631) that rule out roles for Cys-191 (corresponding with the proton donor, Tyr-196, in the structurally related OYE1 enzyme) and His-186 as proton donors, we infer solvent is the source of the proton in the oxidative half-reaction of MR. We demonstrate a key role for Thr-32 in modulating the reduction potential of the FMN, which is decreased approximately 50 mV in the T32A mutant MR. This effects a change in rate-limiting step in the catalytic cycle of the T32A enzyme with the oxidizing substrate 2-cyclohexenone. Despite the conservation of Trp-106 throughout the OYE family, we show this residue does not play a major role in catalysis, although affects on substrate and coenzyme binding are observed in a W106F enzyme. Our studies show some similarities, but also major differences, in the catalytic mechanism of MR and OYE1, and emphasize the need for caution in inferring mechanism by structural comparison of highly related enzymes in the absence of solution studies.
Journal of Biological Chemistry 08/2005; 280(29):27103-10. · 4.77 Impact Factor
