Localization by site-directed mutagenesis of a galantamine binding site on α7 nicotinic acetylcholine receptor extracellular domain.

Galantamine is an approved drug treatment for Alzheimer's disease. Initially identified as a weak cholinesterase inhibitor, we have established that galantamine mainly acts as an 'allosterically potentiating ligand (APL)' of nicotinic acetylcholine receptors (nAChR). Meanwhile other 'positive allosteric modulators (PAM)' of nAChR channel activity have been discovered, and for one of them a binding site within the transmembrane domain has been proposed. Here we show, by performing site-directed mutagenesis studies of ectopically expressed chimeric chicken α7/mouse 5-hydroxytryptamine 3 receptor-channel complex, in combination with whole-cell current measurements, in the presence and absence of galantamine, that the APL binding site is different from the proposed PAM binding site. We demonstrate that residues T197, I196, and F198 of ß-strand 10 represent major elements of the galantamine binding site. Residue K123, earlier suggested as being 'close to' the APL binding site, is not part of this site but rather appears to play a role in coupling of agonist binding to channel opening and closing. Our data confirm our earlier results that the galantamine binding site is different from the ACh binding site. Both sites are in close proximity and hence may influence each other in a synergistic fashion. Other interesting areas identified in the present study are a 'hinge' region around and containing residues F122, K123, and K143 possibly being involved in relaying the signal of agonist binding to gating of the transmembrane channel, and a 'folding centre', with P119 as the dominating residue, that crucially positions the agonist binding site with respect to the hinge region.

Structural model for the binding sites of allosterically potentiating ligands on nicotinic acetylcholine receptors.

Current treatments of Alzheimer's disease include the allosteric potentiation of nicotinic acetylcholine receptor (nAChR) response. The location of the binding site for allosteric potentiating ligands (APLs) within the receptor is not yet fully understood. Based on homology models for the ligand binding domain of human alpha7, human alpha4beta2, and chicken alpha7 receptors, as well as blind docking experiments with galanthamine, physostigmine, codeine, and 5HT, we identified T197 as an essential element of the APL binding site at the outer surface of the ligand binding domain (LBD) of nAChR. We also found the previously known galanthamine binding site in the region of K123 at the inside of the receptor funnel, which, however, was shown to not be part of the APL site. Our results are verified by site-directed mutagenesis and electrophysiological experiments, and suggest that APL and ACh bind to different sites on nicotinic receptors and that allosteric potentiation may arise from a direct interplay between both these sites.

Probing the nanosecond dynamics of a designed three-stranded beta-sheet with a massively parallel molecular dynamics simulation.

Recently a temperature-jump FTIR study of a designed three-stranded sheet showing a fast relaxation time of approximately 140 +/- 20 ns was published. We performed massively parallel molecular dynamics simulations in explicit solvent to probe the structural events involved in this relaxation. While our simulations produce similar relaxation rates, the structural ensemble is broad. We observe the formation of turn structure, but only very weak interaction in the strand regions, which is consistent with the lack of strong backbone-backbone NOEs in previous structural NMR studies. These results suggest that either (D)P(D)P-II folds at time scales longer than 240 ns, or that (D)P(D)P-II is not a well-defined three-stranded beta-sheet. This work also provides an opportunity to compare the performance of several popular forcefield models against one another.

Accelerating molecular dynamic simulation on the cell processor and Playstation 3.

Implementation of molecular dynamics (MD) calculations on novel architectures will vastly increase its power to calculate the physical properties of complex systems. Herein, we detail algorithmic advances developed to accelerate MD simulations on the Cell processor, a commodity processor found in PlayStation 3 (PS3). In particular, we discuss issues regarding memory access versus computation and the types of calculations which are best suited for streaming processors such as the Cell, focusing on implicit solvation models. We conclude with a comparison of improved performance on the PS3's Cell processor over more traditional processors.

Effects of long-range electrostatic forces on simulated protein folding kinetics.

Molecular dynamics simulations are a useful tool for characterizing protein folding pathways. There are several methods of treating electrostatic forces in these simulations with varying degrees of physical fidelity and computational efficiency. In this article, we compare the reaction field (RF) algorithm, particle-mesh Ewald (PME), and tapered cutoffs with increasing cutoff radii to address the impact of the electrostatics method employed on the folding kinetics. We quantitatively compare different methods by a correlation of quantitative measures of protein folding kinetics. The results of these comparisons show that for protein folding kinetics, the RF algorithm can quantitatively reproduce the kinetics of the more costly PME algorithm. These results not only assist the selection of appropriate algorithms for future simulations, but also give insight on the role that long-range electrostatic forces have in protein folding.

Time resolved structure analysis of growing beta-amyloid fibers.

Formation of beta-amyloid plaques is a crucial feature of Alzheimer's disease. In the present work time resolved static light scattering was applied to investigate the size and shape of growing beta-amyloid aggregates preceding plaque formation. The beta-amyloid protein with 40 amino acid residues was used. Salt free buffer solutions and solutions with 0.15M NaCl at 37 degrees C served as the aggregation medium. The focus lay on the first 2h following initiation of the aggregation process which corresponds to the protofibril phase. Addition of the NaCl accelerated the aggregation process considerably. Scattering data from aggregation in saline solutions indicated formation of long fibers which suggest interpretation of data with the worm-like chain model. Two important results were revealed: (i) At the end of the time resolved recordings, the worm-like chain model provided a fully adequate picture for the growing aggregates. Chain stiffness is characterised in terms of the persistence length, which is close to 50 nm. The linear mass density of the growing fibers approached a value of two monomers per nm corresponding to single stranded fibers, which is in accordance with presently existing models for the aggregation of beta-amyloid. The fibers finally reached contour lengths of several thousand nanometers. (ii) The plateau values for the persistence length and linear mass density observed in the final regime are gradually approached from higher values. This observation is inconsistent with simple worm-like chains. Rather does it indicate existence of another species during the initial phase of the aggregation, in addition to monomers and fibers. Aside from further insight into fundamental aspects of beta-amyloid aggregation, time resolved static light scattering provides an appropriate tool for assay tests with drugs designed to interfere with the aggregation process.

The Chemistry Development Kit (CDK): an open-source Java library for Chemo- and Bioinformatics.

The Chemistry Development Kit (CDK) is a freely available open-source Java library for Structural Chemo- and Bioinformatics. Its architecture and capabilities as well as the development as an open-source project by a team of international collaborators from academic and industrial institutions is described. The CDK provides methods for many common tasks in molecular informatics, including 2D and 3D rendering of chemical structures, I/O routines, SMILES parsing and generation, ring searches, isomorphism checking, structure diagram generation, etc. Application scenarios as well as access information for interested users and potential contributors are given.

Galanthamine as bis-functional ligand for the acetylcholinesterase.

Acetylcholinesterase plays a key role in the development of Alzheimer's disease as this enzyme is responsible for cleavage of the neurotransmitter acetylcholine, and, according to recent investigations, also promotes aggregation of beta-amyloid peptides, which causes plaque formation in synaptic areas. We have performed a molecular modeling study to investigate bis-galanthamine derivatives connected by a methylene spacer of varying length as dual acting acetylcholinesterase ligands. Our results suggest that such ligands indeed can interact simultaneously with both biological functions of the enzyme and should therefore serve as the basis for a further development of bis-functional Alzheimer drugs.