Exploring the origin of the ion selectivity of the KcsA potassium channel

The availability of structural information about biological ion channels provides an opportunity to gain a detailed understanding of the control of ion selectivity by biological systems. However, accomplishing this task by computer simulation approaches is very challenging. First, although the activation barriers for ion transport can be evaluated by microscopic simulations, it is hard to obtain accurate results by such approaches. Second, the selectivity is related to the actual ion current and not directly to the individual activation barriers. Thus, it is essential to simulate the ion currents and this cannot be accomplished at present by microscopic MD approaches. In order to address this challenge, we developed and refined an approach capable of evaluating ion current while still reflecting the realistic features of the given channel. Our method involves generation of semimacroscopic free energy surfaces for the channel/ions system and Brownian dynamics (BD) simulations of the corresponding ion current. In contrast to most alternative macroscopic models, our approach is able to reproduce the difference between the free energy surfaces of different ions and thus to address the selectivity problem. Our method is used in a study of the selectivity of the KcsA channel toward the K+ and Na+ ions. The BD simulations with the calculated free energy profiles produce an appreciable selectivity. To the best of our knowledge, this is the first time that the trend in the selectivity in the ion current is produced by a computer simulation approach. Nevertheless, the calculated selectivity is still smaller than its experimental estimate. Recognizing that the calculated profiles are not perfect, we examine how changes in these profiles can account for the observed selectivity. It is found that the origin of the selectivity is more complex than generally assumed. The observed selectivity can be reproduced by increasing the barrier at the exit and the entrance of the selectivity filter, but the necessary changes in the barrier approach the limit of the error in the PDLD/S-LRA calculations. Other options that can increase the selectivity are also considered, including the difference between the Na+...Na+ and K+...K+ interaction. However, this interesting effect does not appear to lead to a major difference in selectivity since the Na+ ions at the limit of strong interaction tend to move in a less concerted way than the K+ ions. Changes in the relative binding energies at the different binding sites are also not so effective in changing the selectivity. Finally, it is pointed out that using the calculated profiles as a starting point and forcing the model to satisfy different experimentally based constraints, should eventually provide more detailed understanding of the different complex factors involved in ion selectivity of biological channels.     

Solution structure of the fibronectin type III domain from Bacillus circulans WL-12 chitinase A1

Growing evidence suggests that horizontal gene transfer plays an integral role in the evolution of bacterial genomes. One of the debated examples of horizontal gene transfer from animal to prokaryote is the fibronectin type III domain (FnIIID). Certain extracellular proteins of soil bacteria contain an unusual cluster of FnIIIDs, which show sequence similarity to those of animals and are likely to have been acquired horizontally from animals. Here we report the solution structure of the FnIIID of chitinase A1 from Bacillus circulans WL-12. To the best of our knowledge, this is the first tertiary structure to be reported for an FnIIID from a bacterial protein. The structure of the domain shows significant similarity to FnIIIDs from animal proteins. Sequence comparisons with FnIIIDs from other soil bacteria proteins show that the core-forming residues are highly conserved and, thus, are under strong evolutionary pressure. Striking similarities in the tertiary structures of bacterial FnIIIDs and their mammalian counterparts may support the hypothesis that the evolution of the FnIIID in bacterial carbohydrases occurred horizontally. The total lack of surface-exposed aromatic residues also suggests that the role of this FnIIID is different from those of other bacterial beta-sandwich domains, which function as carbohydrate-binding modules.     

The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization

Synaptotagmin VII (Syt VII), a proposed regulator for Ca2+-dependent exocytosis, showed a robust Ca2+-dependent oligomerization property via its two C2 domains (Fukuda, M., and Mikoshiba, K. (2001) J. Biol. Chem. 276, 27670-27676), but little is known about its structure or the critical residues directly involved in the oligomerization interface. In this study, site-directed mutagenesis and chimeric analysis between Syt I and Syt VII showed that three Asp residues in Ca2+-binding loop 1 or 3 (Asp-172, Asp-303, and Asp-357) are crucial to robust Ca(2+)-dependent oligomerization. Unlike Syt I, however, the polybasic sequence in the beta4 strands of the C2 structures (so-called "C2 effector domain") is not involved in the Ca2+-dependent oligomerization of Syt VII. The results also showed that the Ca2+-binding loops of the two C2 domains cooperatively mediate Syt VII oligomerization (i.e. the presence of redundant Ca2+-binding site(s)) as well as the importance of Ca2+-dependent oligomerization of Syt VII in Ca2+-regulated secretion. Expression of wild-type tandem C2 domains of Syt VII in PC12 cells inhibited Ca2+-dependent neuropeptide Y release, whereas mutant fragments lacking Ca2+-dependent oligomerization activity had no effect. Finally, rotary-shadowing electron microscopy showed that the Ca2+-dependent oligomer of Syt VII is "a large linear structure," not an irregular aggregate. By contrast, in the absence of Ca2+ Syt VII molecules were observed to form a globular structure. Based on these results, we suggest that the linear Ca2+-dependent oligomer may be aligned at the fusion site between vesicles and plasma membrane and modulate Ca2+-regulated exocytosis by opening or dilating fusion pores.     

Direct, Ca2+-dependent interaction between tubulin and synaptotagmin I: a possible mechanism for attaching synaptic vesicles to microtubules

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin I. The binding depended on mm Ca(2+), and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 microm tubulin. The Ca(2+) dependence mainly resulted from Ca(2+) binding to the Ca(2+) ligands of synaptotagmin I. The C-terminal region of beta-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca(2+). These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca(2+) concentrations.     

Generation of amyloid beta protein from a presenilin-1 and betaAPP complex

Presenilin-1 (PS1) is a causative gene in early onset familial Alzheimer's disease (FAD). FAD-linked mutant PS1s significantly increased Abeta40 and Abeta42(43) levels (P < 0.001) and decreased the production of an 11.4 kD (beta-stub) and an 8.7 kD (alpha-stub) carboxyl-terminal fragment of amyloid beta precursor protein (betaAPP-CTFs) (P < 0.01). In the 2% CHAPS extracted lysates, the complex containing the amino-terminal fragment of PS1 (PS1-NTF), the carboxyl-terminal fragments of PS1 (PS1-CTF), and betaAPP-CTFs was identified. Incubation of this isolated complex at pH 6.4 showed the direct generation of Abeta40 and gamma-stub from this complex. This reaction was inhibited by a gamma-secretase inhibitor. The degrading rate of a co-precipitated beta-stub was facilitated under the presence of FAD-linked mutant PS1s. This findings suggest that the direct generation of Abeta from the complex may play an important role in the pathogenesis of Alzheimer's disease.     

An enzymatic fluorometric assay for pyridoxal with high specificity and sensitivity

An enzymatic fluorometric assay for pyridoxal with pyridoxal dehydrogenase was developed. The detection limit was about 10 pmol: the calibration curve of pyridoxal showed high linearity (r=0.993). The values obtained by this method correlated well with those by the HPLC method. The enzyme had a high specificity for pyridoxal, and thus animal samples could be directly analyzed without separation of pyridoxal 5'-phosphate by column chromatography.     

An efficient chemical synthesis of nicotinamide riboside (NAR) and analogues

A simple and efficient synthesis of nicotinamide riboside (NAR) 1 and derivatives 4 and 5 via trimethylsilyl trifluoromethanesulfonate (TMSOTf)-mediated N-glycosilation followed by spontaneous deacetylation by treating with methanol is reported.     

Prediction of biologically significant components from microarray data: Independently Consistent Expression Discriminator (ICED)

MOTIVATION: Class distinction is a supervised learning approach that has been successfully employed in the analysis of high-throughput gene expression data. Identification of a set of genes that predicts differential biological states allows for the development of basic and clinical scientific approaches to the diagnosis of disease. The Independent Consistent Expression Discriminator (ICED) was designed to provide a more biologically relevant search criterion during predictor selection by embracing the inherent variability of gene expression in any biological state. The four components of ICED include (i) normalization of raw data; (ii) assignment of weights to genes from both classes; (iii) counting of votes to determine optimal number of predictor genes for class distinction; (iv) calculation of prediction strengths for classification results. The search criteria employed by ICED is designed to identify not only genes that are consistently expressed at one level in one class and at a consistently different level in another class but identify genes that are variable in one class and consistent in another. The result is a novel approach to accurately select biologically relevant predictors of differential disease states from a small number of microarray samples. RESULTS: The data described herein utilized ICED to analyze the large AML/ALL training and test data set (Golub et al., 1999, Science, 286, 531-537) in addition to a smaller data set consisting of an animal model of the childhood neurodegenerative disorder, Batten disease, generated for this study. Both of the analyses presented herein have correctly predicted biologically relevant perturbations that can be used for disease classification, irrespective of sample size. Furthermore, the results have provided candidate proteins for future study in understanding the disease process and the identification of potential targets for therapeutic intervention.