Publication date: Available online 14 November 2015
Source:Journal of Molecular Biology

Author(s): Nicholas Furnham, Natalie L. Dawson, Syed A. Rahman, Janet M. Thornton, Christine A. Orengo

Enzymes, as biological catalysts, form the basis of all forms of life. How these proteins have evolved their functions remains a fundamental question in biology. Over 100years of detailed biochemistry studies, combined with the large volumes of sequence and protein structural data now available, means we are able to perform large-scale analyses to address this question. Using a range of computational tools and resources we have compiled information on all experimentally annotated changes in enzyme function within 379 structurally defined protein domain superfamilies, linking the changes observed in functions during evolution, to changes in reaction chemistry. Many superfamilies show changes in function at some level, although one function often dominates one superfamily. We use quantitative measures of changes in reaction chemistry to reveal the various types of chemical changes occurring during evolution and exemplify these by detailed examples. Additionally, we use structural information of the enzymes active site to examine how different superfamilies have changed their catalytic machinery during evolution. Some superfamilies have changed the reactions they perform without changing catalytic machinery. In others large changes of enzyme function, both in terms of overall chemistry and substrate specificity, have been brought about by significant changes in catalytic machinery. Interestingly, in some superfamilies’ relatives perform similar functions but with different catalytic machineries. This analysis highlights characteristics of functional evolution across a wide range of superfamilies’, providing insights that will be useful in predicting the function of uncharacterized sequences as well as the design of new synthetic enzymes.
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Publication date: Available online 14 November 2015
Source:Journal of Molecular Biology

Author(s): Yosuke Nishikawa, Momoko Inatomi, Haruka Iwasaki, Genji Kurisu

Dynein is a large microtubule-based motor complex that requires tight coupling of intra-molecular ATP hydrolysis with the generation of mechanical force and track-binding activity. However, the microtubule-binding domain is structurally separated by about 15nm from the nucleotide-binding sites by a coiled-coil stalk. Thus, long-range two-way communication is necessary for coordination between the catalytic cycle of ATP hydrolysis and dynein's track-binding affinities. To investigate the structural changes that occur in the dynein stalk region to produce two different microtubule affinities, here we improve the resolution limit of the previously reported structure of the entire stalk region and we investigate structural changes in the dynein stalk and strut/buttress regions by comparing currently available X-ray structures. In the light of recent crystal structures, the basis of the transition from the low-affinity to the high-affinity coiled-coil registry is discussed. A concerted movement model previously reported by Carter and Vale is modified more specifically, and we proposed it as the open zipper model.
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Publication date: Available online 14 November 2015
Source:Journal of Molecular Biology

Author(s): Rosanne L.L. Hill, Terje Dokland

Staphylococcus aureus pathogenicity islands (SaPIs) are genetic elements that are mobilized by specific helper phages. The initial step in mobilization is the derepression of the SaPI by the interaction of a phage protein with the SaPI master repressor Stl. Stl proteins are highly divergent between different SaPIs and respond to different phage-encoded derepressors. One such SaPI, SaPIbov1, is derepressed by the dUTPase (Dut) of bacteriophage 80α (Dut80α) and its phage ϕ11 homolog, Dut11. We previously showed that SaPIbov1 could also be mobilized by phage ϕNM1, even though its dut gene is not homologous with that of 80α. Here, we show that ϕNM1 dut encodes a type 2 dUTPase (DutNM1), which has an α-helical structure that is distinct from the type 1 trimeric, β-sheet structure of Dut80α. Deletion of dut NM1 abolishes the ability of ϕNM1 to mobilize SaPIbov1. Like Dut80α, DutNM1 forms a direct interaction with SaPIbov1 Stl both in vivo and in vitro, leading to inhibition of the dUTPase activity and Stl release from its target DNA. This work provides novel insights into the diverse mechanisms of genetic mobilization in S. aureus.
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Publication date: Available online 14 November 2015
Source:Journal of Molecular Biology

Author(s): Minoru Kanehisa, Yoko Sato, Kanae Morishima

BlastKOALA and GhostKOALA are automatic annotation servers for genome and metagenome sequences, which perform KO (KEGG Orthology) assignments to characterize individual gene functions and reconstruct KEGG pathways, BRITE hierarchies and KEGG modules to infer high-level functions of the organism or the ecosystem. Both servers are made freely available at the KEGG Web site (http://www.kegg.jp/blastkoala/). In BlastKOALA, the KO assignment is performed by a modified version of the internally used KOALA algorithm after the BLAST search against a non-redundant dataset of pangenome sequences at the species, genus or family level, which is generated from the KEGG GENES database by retaining the KO content of each taxonomic category. In GhostKOALA, which utilizes more rapid GHOSTX for database search and is suitable for metagenome annotation, the pangenome dataset is supplemented with Cd-hit clusters including those for viral genes. The result files may be downloaded and manipulated for further KEGG Mapper analysis, such as comparative pathway analysis using multiple BlastKOALA results.
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Publication date: Available online 12 November 2015
Source:Journal of Molecular Biology

Author(s): Charles Sindelar, Andrew Huehn

Publication date: Available online 12 November 2015
Source:Journal of Molecular Biology

Author(s): Iva Lučić, Linda Truebestein, Thomas A. Leonard

Diacylglycerol (DAG) activates the eight conventional and novel isozymes of protein kinase C (PKC) by binding to their C1 domains. The crystal structure of PKCβII in a partially activated conformation showed how the C1B domain regulates activity by clamping a helix in the C-terminal AGC extension of the kinase domain. Here we show that the global three-dimensional shape of the conventional and novel PKCs is conserved despite differences in the order of the domains in their primary sequences. The membrane translocation phenotypes of mutants in the C1B clamp are consistent across all DAG-activated PKCs, demonstrating conservation of this regulatory interface. We now identify a novel interface that sequesters the C1A domain in PKCβII in a membrane-inaccessible state and we generalize this to all DAG-activated PKCs. In the conventional PKCs, we identify a novel element of their C2 domains that additionally contributes to the stability of the inactive conformation. We demonstrate that the interdomain linkers play important roles in permitting and stabilizing this state. We propose a multi-step activation mechanism in which the sequential and cooperative binding of the regulatory domains to the membrane is coupled to allosteric activation of the kinase domain by DAG and that acquisition of full catalytic activity requires DAG binding to the C1B domain. In light of the conservation of shape and intramolecular architecture, we propose that this mechanism is common to all DAG-activated PKCs.
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Publication date: Available online 11 November 2015
Source:Journal of Molecular Biology

Author(s): Ali R. Zomorrodi, Daniel Segrè

As the indispensable role of natural microbial communities in many aspects of life on Earth is uncovered, the bottom-up engineering of synthetic microbial consortia with novel functions is becoming an attractive alternative to engineering single-species systems. Here, we summarize recent work on synthetic microbial communities with a particular emphasis on open challenges and opportunities in environmental sustainability and human health. We next provide a critical overview of mathematical approaches, ranging from phenomenological to mechanistic, to decipher the principles that govern the function, dynamics and evolution of microbial ecosystems. Finally, we present our outlook on key aspects of microbial ecosystems and synthetic ecology that require further developments, including the need for more efficient computational algorithms, a better integration of empirical methods and model-driven analysis, the importance of improving gene function annotation, and the value of a standardized library of well-characterized organisms to be used as building blocks of synthetic communities.
Graphical abstract

Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Madhurima Das, Christopher J. Wilson, Xiaohu Mei, Thomas Wales, John R. Engen, Olga Gursky

ApoA-I, the major protein of plasma high-density lipoprotein, removes cellular cholesterol and protects against atherosclerosis. ApoA-I mutations can cause familial amyloidosis, a life-threatening disease wherein N-terminal protein fragments form fibrils in vital organs. To unveil the protein misfolding mechanism and to understand why some mutations cause amyloidosis while others do not, we analyzed the structure, stability and lipid-binding properties of naturally occurring mutants of full-length human apoA-I causing either amyloidosis (G26R, W50R, F71Y, L170P) or aberrant lipid metabolism (L159R). Global and local protein conformation and dynamics in solution were assessed by circular dichroism, fluorescence, and hydrogen-deuterium exchange mass spectrometry. All mutants showed increased deuteration in residues 14-22, supporting our hypothesis that decreased protection of this major amyloid “hot spot” can trigger protein misfolding. In addition, L159R showed local helical unfolding near the mutation site, consistent with cleavage of this mutant in plasma to generate the labile 1-159 fragment. Together, the results suggest that reduced protection of the major amyloid “hot spot”, combined with structural integrity of the native helix-bundle conformation, shift the balance from protein clearance to β-aggregation. A delicate balance between the overall structural integrity of a globular protein and the local destabilization of its amyloidogenic segments may be a fundamental determinant of this and other amyloid diseases. Furthermore, mutation-induced conformational changes observed in the helix bundle, which comprises N-terminal 75% of apoA-I, and its flexible C-terminal tail suggest the propagation of structural perturbations to distant sites via an unexpected template-induced ensemble-based mechanism, challenging the classical structure-based view.
Graphical abstract

Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Raffaela Bung, Philipp Wörsdörfer, Marc Christian Thier, Kathrin Vogt, Martina Gebhardt, Frank Edenhofer

Direct cell conversion developed into an important paradigm for generating cells with enhanced differentiation capability. We combined a transcription factor-based cell fate conversion strategy with the use of pharmacological compounds to derive early neuroepithelial progenitor cells from developmentally more restricted radial glia type neural stem cells (RG-NSCs). By combining the small molecules CHIR99021, Tranylcypromine, SB431542 and valproic acid with viral transduction of the transcription factors c-Myc and the POU domain transcription factor Brn2 we dedifferentiated RG-NSCs into an early neuroepithelial progenitor cell state within 6days. RT-PCR analyses showed a rapid down-regulation of the radial glia markers Olig2 and Vimentin during conversion, whereas the neuroepithelial markers Dach1 and Sox1 were fastly up-regulated. Furthermore, a switch from N- to E-Cadherin indicates a mesenchymal-to-epithelial transition. The differentiation of cells converted by Brn-2/c-Myc yielded smooth muscle actin- and Peripherin-positive cells in addition to the neuronal marker TUJ1 and cells that are positive for the glial marker GFAP. This differentiation potential suggests that the applied reprogramming strategy induced an early neuroepithelial cell population, which might resemble cells of the neural border or even more primitive neuroepithelial cells.
Graphical abstract

Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Vijay kumar Srivastava, Mintu Chandra, Yumiko Saito-Nakano, Tomoyoshi Nozaki, Sunando Datta

The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. EhRabX3 is a catalytically inefficient amoebic Rab protein, which is unique among the eukaryotic Ras superfamily by virtue of its tandem domain organization. Here, we report the crystal structures of GDP-bound fast hydrolyzing mutant (V71A/K73Q) and GTP-bound wild type EhRabX3 at 3.1 and 2.8 Å resolutions, respectively. Though both G-domains possess “P-loop containing nucleoside triphosphate hydrolases fold”, only the NTD binds to guanine nucleotide. The relative orientation of the NTD and CTD is stabilized by numerous inter-domain interactions. Compared to other Ras superfamily members, both the GTPase domains displayed large deviation in switch II, perhaps due to non-conservative substitutions in this region. As a result, entire switch II is restructured and moved away from the nucleotide binding pocket, providing a rationale for the diminished GTPase activity of EhRabX3. The N-terminal GTPase domain possesses unusually large number of cysteine residues. X-ray crystal structure of the fast hydrolyzing mutant of EhRabX3 revealed that C39 and C163 formed an intra-molecular disulfide bond. Subsequent mutational and biochemical studies suggest that C39 and C163 are critical for maintaining the structural integrity and function of EhRabX3. Structure-guided functional investigation of cysteine mutants could provide the physiological implications of the disulfide bond and allow us to design potential inhibitors for the better treatment of intestinal amebiasis.
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Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Nicole Monroe, Christopher P. Hill

Meiotic clade AAA ATPases, which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin, and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT pathway, and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops, and that recruitment to the protein lattice via MIT domains also relieves auto-inhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule-binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, and potentially katanin and fidgetin, are highly coordinated.
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Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Lucile Moynié, Anthony G. Hope, Kara Finzel, Jason Schmidberger, Stuart M. Leckie, Gunter Schneider, Michael D. Burkart, Andrew D. Smith, David W. Gray, James H. Naismith

Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 =5.7±0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.
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Publication date: Available online 7 November 2015
Source:Journal of Molecular Biology

Author(s): Eugenia Voziyanova, Rachelle P. Anderson, Riddhi Shah, Feng Li, Yuri Voziyanov

Genome engineering benefits from the availability of DNA modifying enzymes that have different target specificities and have optimized performance in different cell types. This variety of site-specific enzymes can be used to develop complex genome engineering applications at multiple loci. Although eight yeast site-specific tyrosine recombinases are known, only Flp is actively used in genome engineering. To expand the pool of the yeast site-specific tyrosine recombinases capable of mediating genome manipulations in mammalian cells, we engineered and analyzed variants of two tyrosine recombinases: R and TD. The activity of the evolved variants, unlike the activity of the native R and TD recombinases, is suitable for genome engineering in Escherichia coli and mammalian cells. Unexpectedly, we found that R recombinase benefits from the shortening of its C-terminus. We also found that the activity of wild-type R can be modulated by its non-consensus “head” sequence but this modulation became not apparent in the evolved R variants. The engineered recombinase variants were found to be active in all recombination reactions tested: excision, integration, and dual recombinase-mediated cassette exchange. The analysis of the latter reaction catalyzed by the R/TD recombinase pair shows that the condition supporting the most efficient replacement reaction favors efficient TD-mediated integration reaction while favoring efficient R-mediated integration and deletion reactions.
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Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22

Next week, the United Nations Climate Change Conference, COP21, will be held in Paris. The goal is to achieve an international agreement to stem climate change—in particular, an agreement on how to keep global warming below a 2°C rise, or less, over preindustrial levels. As the newly elected chair of the Intergovernmental Panel on Climate Change (IPCC), I am hopeful that an agreement will be reached that builds a more sustainable, prosperous world. Author: Hoesung Lee

In science news around the world, the current El Niño is shaping up to be among the three biggest on record, U.S. and Cuban science agencies team up to manage and study marine protected areas, a letter signed by 26 wildlife scientists urges the U.S. Department of the Interior to take gray wolves off the endangered species list, Europe's first Tyrannosaurus rex skeleton is being prepped to go on display next month in Berlin, and more. Also, Nobel laureate in medicine Elizabeth Blackburn has been named the new head of the Salk Institute for Biological Sciences, and President Obama awards research mathematician and space pioneer Katherine Johnson the Presidential Medal of Freedom. And a dance-off between competing interests over water resources wins Science's 2015 Dance Your Ph.D. contest.

Managers of the troubled ITER fusion project have announced a new schedule that is likely to push the estimated date of completion back by 6 years, to 2025, and add roughly €2 billion to the project's ballooning cost. The changes, presented at a meeting of ITER's governing council, resulted from a comprehensive review that ITER's director-general ordered earlier this year. In response, the project's international partners—China, the European Union, India, Japan, Russia, South Korea, and the United States—said they plan to carry out an independent review, looking for ways to tighten the schedule and costing, and have put off approving the baseline until the next council meeting in 6 months. Author: Daniel Clery

A little-known virus called Zika has caused outbreaks in Pacific Ocean islands the past few years and has arrived in South America this year. Scientists predict it will spread far and wide in the Western Hemisphere, and perhaps in southern Europe as well, because the Aedes mosquitoes that transmit the virus are so widespread. Scientifically speaking, Zika virus is still largely terra incognita. Its symptoms, including rash, fatigue, headaches, muscle pains, and swollen and painful joints, appear to be generally mild, but during an outbreak in French Polynesia that started in 2013, some patients developed a serious neurological condition named Guillain-Barré syndrome. Although it is primarily spread by mosquitoes, some evidence suggests sexual transmission is possible as well. Author: Martin Enserink

Last week, the U.S. National Institutes of Health (NIH) announced that it is ending its support for invasive research on chimpanzees. NIH Director Francis Collins said that a colony of 50 chimps it had planned to keep in reserve for research—after retiring the rest—is no longer needed. NIH also made clear that it will no longer fund invasive studies on any other chimps. The move pleased groups that have pushed to end the use of chimpanzees in biomedical research. But some researchers expressed disappointment, noting that the colony was intended to be available in case chimps were needed as a research model in the future. Author: Jocelyn Kaiser