Structural and Biophysical Characterization of the Syk Activation Switch

Syk is an essential non-receptor tyrosine kinase in intracellular immunological signaling, and the control of Syk kinase function is considered as a valuable target for pharmacological intervention in autoimmune or inflammation diseases. Upon immune receptor stimulation, the kinase activity of Syk is regulated by binding of phosphorylated immune receptor tyrosine-based activating motifs (pITAMs) to the N-terminal tandem Src homology 2 (tSH2) domain and by autophosphorylation with consequences for the molecular structure of the Syk protein. Here, we present the first crystal structures of full-length Syk (fl-Syk) as wild type and as Y348F,Y352F mutant forms in complex with AMP-PNP revealing an autoinhibited conformation. The comparison with the crystal structure of the truncated Syk kinase domain in complex with AMP-PNP taken together with ligand binding studies by surface plasmon resonance (SPR) suggests conformational differences in the ATP sites of autoinhibited and activated Syk forms. This hypothesis was corroborated by studying the thermodynamic and kinetic interaction of three published Syk inhibitors with isothermal titration calorimetry and SPR, respectively. We further demonstrate the modulation of inhibitor binding affinities in the presence of pITAM and discuss the observed differences of thermodynamic and kinetic signatures. The functional relevance of pITAM binding to fl-Syk was confirmed by a strong stimulation of in vitro autophosphorylation. A structural feedback mechanism on the kinase domain upon pITAM binding to the tSH2 domain is discussed in analogy of the related family kinase ZAP-70 (Zeta-chain-associated protein kinase 70). Surprisingly, we observed distinct conformations of the tSH2 domain and the activation switch including Tyr348 and Tyr352 in the interdomain linker of Syk in comparison to ZAP-70.     

Evidence for Benzylsuccinate Synthase Subtypes Obtained by Using Stable Isotope Tools

We studied the benzylsuccinate synthase (Bss) reaction mechanism with respect to the hydrogen-carbon bond cleavage at the methyl group of toluene by using different stable isotope tools. Λ values (slopes of linear regression curves for carbon and hydrogen discrimination) for two-dimensional compound-specific stable isotope analysis (2D-CSIA) of toluene activation by Bss-containing cell extracts (in vitro studies) were found to be similar to previously reported data from analogous experiments with whole cells (in vivo studies), proving that Λ values generated by whole cells are caused by Bss catalysis. The Bss enzymes of facultative anaerobic bacteria produced smaller Λ values than those of obligate anaerobes. In addition, a partial exchange of a single deuterium atom in benzylsuccinate with hydrogen was observed in experiments with deuterium-labeled toluene. In this study, the Bss enzymes of the tested facultative anaerobes showed 3- to 8-fold higher exchange probabilities than those for the enzymes of the tested obligate anaerobic bacteria. The phylogeny of the Bss variants, determined by sequence analyses of BssA, the gene product corresponding to the α subunit of Bss, correlated with the observed differences in Λ values and hydrogen exchange probabilities. In conclusion, our results suggest subtle differences in the reaction mechanisms of Bss isoenzymes of facultative and obligate anaerobes and show that the putative isoenzymes can be differentiated by 2D-CSIA.     

Cyclohexanecarboxyl-Coenzyme A (CoA) and Cyclohex-1-ene-1-Carboxyl-CoA Dehydrogenases,Two Enzymes Involved in the Fermentation of Benzoate and Crotonate in Syntrophus aciditrophicus

The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.     

Mosaics in the mangroves: allopatric diversification of tree‐climbing mudwhelks (Gastropoda: Potamididae: Cerithidea) in the Indo‐West Pacific

The Indo‐Australian Archipelago (IAA) is the richest area of biodiversity in the marine realm, yet the processes that generate and maintain this diversity are poorly understood and have hardly been studied in the mangrove biotope. Cerithidea is a genus of marine and brackish‐water snails restricted to mangrove habitats in the Indo‐West Pacific, and its species are believed to have a short pelagic larval life. Using molecular and morphological techniques, we demonstrate the existence of 15 species, reconstruct their phylogeny and plot their geographical ranges. Sister species show a pattern of narrowly allopatric ranges across the IAA, with overlap only between clades that show evidence of ecological differentiation. These allopatric mosaic distributions suggest that speciation may have been driven by isolation during low sea‐level stands, during episodes preceding the Plio‐Pleistocene glaciations. The Makassar Strait forms a biogeographical barrier hindering eastward dispersal, corresponding to part of Wallace's Line in the terrestrial realm. Areas of maximum diversity of mangrove plants and their associated molluscs do not coincide closely. © 2013 The Natural History Museum. Biological Journal of the Linnean Society © 2013 The Linnean Society of London, 2013, 110 , 564–580.     

Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3

Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.     

Structural and biochemical characterization of Rv2140c,a phosphatidylethanolamine-binding protein from Mycobacterium tuberculosis

Rv2140c is one of many conserved Mycobacterium tuberculosis proteins for which no molecular function has been identified. We have determined a high-resolution crystal structure of the Rv2140c gene product, which reveals a dimeric complex that shares strong structural homology with the phosphatidylethanolamine-binding family of proteins. Rv2140c forms low-millimolar interactions with a selection of soluble phosphatidylethanolamine analogs, indicating that it has a role in lipid metabolism. Furthermore, the small molecule locostatin binds to the Rv2140c ligand-binding site and also inhibits the growth of the model organism Mycobacterium smegmatis.     

Structure and Topology of the Huntingtin 1–17 Membrane Anchor by a Combined Solution and Solid-State NMR Approach

The very amino-terminal domain of the huntingtin protein is directly located upstream of the protein’s polyglutamine tract, plays a decisive role in several important properties of this large protein and in the development of Huntington’s disease. This huntingtin 1–17 domain is on the one hand known to markedly increase polyglutamine aggregation rates and on the other hand has been shown to be involved in cellular membrane interactions. Here, we determined the high-resolution structure of huntingtin 1–17 in dodecyl phosphocholine micelles and the topology of its helical domain in oriented phosphatidylcholine bilayers. Using two-dimensional solution NMR spectroscopy the low-energy conformations of the polypeptide were identified in the presence of dodecyl phosphocholine detergent micelles. In a next step a set of four solid-state NMR angular restraints was obtained from huntingtin 1–17 labeled with ¹⁵N and ²H at selected sites. Of the micellar ensemble of helical conformations only a limited set agrees in quantitative detail with the solid-state angular restraints of huntingtin 1–17 obtained in supported planar lipid bilayers. Thereby, the solid-state NMR data were used to further refine the domain structure in phospholipid bilayers. At the same time its membrane topology was determined and different motional regimes of this membrane-associated domain were explored. The pronounced structural transitions of huntingtin 1–17 upon membrane-association result in a α-helical conformation from K6 to F17, i.e., up to the very start of the polyglutamine tract. This amphipathic helix is aligned nearly parallel to the membrane surface (tilt angle ∼77°) and is characterized by a hydrophobic ridge on one side and an alternation of cationic and anionic residues that run along the hydrophilic face of the helix. This arrangement facilitates electrostatic interactions between huntingtin 1–17 domains and possibly with the proximal polyglutamine tract.