The intelligence cycle is dead, long live the intelligence cycle: Rethinking intelligence fundamentals for a new intelligence doctrine

In the spring of 2009 the UK Ministry of Defence elected to undertake a review of the existing military Joint Intelligence Doctrine. The existing doctrine, Joint Warfare Doctrine 2-00 (JWP 2-00) Intelligence Support to Joint Operations had been promulgated in 2003 largely on the basis of coalition-oriented expeditionary and peace support operations in the Balkans, Middle East and Afghanistan. This had replaced an earlier, first edition of JWP 2-00 issued in 1999

The structure of the D49 phospholipase A(2) piratoxin III from Bothrops pirajai reveals unprecedented structural displacement of the calcium-binding loop: possible relationship to cooperative substrate binding

Snake venoms are rich sources of phospholipase A(2) homologues, both active calcium-binding Asp49 enzymes and essentially inactive Lys49 proteins. They are responsible for multiple pharmacological effects, some of which are dependent on catalytic activity and others of which are not. Here, the 2.4 Angstrom X-ray crystal structure of an active Asp49 phospholipase A(2) from the venom of the snake Bothrops pirajai, refined to conventional and free R values of 20.1 and 25.5%, respectively, is reported. Unusually for phospholipases A(2), the dependence of the enzyme rate on the substrate concentration is sigmoidal, implying cooperativity of substrate binding. The unprecedented structural distortion seen for the calcium-binding loop in the present structure may therefore be indicative of a T-state enzyme. An explanation of the interaction between the substrate-binding sites based on the canonical phospholipase A(2) dimer is difficult. However, an alternative putative dimer interface identified in the crystal lattice brings together the calcium-binding loops of neighbouring molecules, along with the C-terminal regions which are disulfide bonded to those loops, thereby offering a possible route of communication between active sites.59225526

The Structure Of The D49 Phospholipase A2 Piratoxin Iii From Bothrops Pirajai Reveals Unprecedented Structural Displacement Of The Calcium-binding Loop: Possible Relationship To Cooperative Substrate Binding

Snake venoms are rich sources of phospholipase A2 homologues, both active calcium-binding Asp49 enzymes and essentially inactive Lys49 proteins. They are responsible for multiple pharmacological effects, some of which are dependent on catalytic activity and others of which are not. Here, the 2.4 Å X-ray crystal structure of an active Asp49 phospholipase A2 from the venom of the snake Bothrops pirajai, refined to conventional and free R values of 20.1 and 25.5%, respectively, is reported. Unusually for phospholipases A2, the dependence of the enzyme rate on the substrate concentration is sigmoidal, implying cooperativity of substrate binding. The unprecedented structural distortion seen for the calcium-binding loop in the present structure may therefore be indicative of a T-state enzyme. An explanation of the interaction between the substrate-binding sites based on the canonical phospholipase A2 dimer is difficult. However, an alternative putative dimer interface identified in the crystal lattice brings together the calcium-binding loops of neighbouring molecules, along with the C-terminal regions which are disulfide bonded to those loops, thereby offering a possible route of communication between active sies.592255262Adams, P.D., Pannu, N.S., Read, R.J., Brünger, A.T., (1997) Proc. Natl Acad. Sci. USA, 94, pp. 5018-5023Arni, R.K., Fontes, M.R., Barberato, C., Gutierrez, J.M., Diaz, C., Ward, R.J., (1999) Arch. Biochem. Biophys., 366, pp. 177-182Barton, G.J., (1993) Protein Eng., 6, pp. 37-40Beghini, D.G., Toyama, M.H., Hyslop, S., Sodek, L.C., Novello, J.C., Marangoni, S., (2000) J. Protein Chem., 19, pp. 679-684Brünger, A.T., (1992) Nature (London), 355, pp. 472-474Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Warren, G.L., (1998) Acta Cryst., D54, pp. 905-921Burke, J.R., Witmer, M.R., Tredup, J., Micanovic, R., Gregor, K.R., Lahiri, J., Tramposch, K.M., Villafranca, J.J., (1995) Biochemistry, 34, pp. 15165-15174Carredano, E., Westerlund, B., Persson, B., Saarinen, M., Ramaswamy, S., Eaker, D., Eklund, H., (1998) Toxicon, 36, pp. 75-92Cha, S.S., Lee, D., Adams, J., Kurdyla, J.T., Jones, C.S., Marshall, L.A., Bolognese, B., Oh, B.H., (1996) J. Med. Chem., 39, pp. 3878-3881Cho, W., Kézdy, F.J., (1991) Methods Enzymol., 197, pp. 75-79(1994) Acta Cryst., D50, pp. 760-763Cowtan, K.D., Zhang, K.Y., (1999) Prog. Biophys. Mol. Biol., 72, pp. 245-270Dessen, A., Tang, J., Schmidt, H., Stahl, M., Clark, J.D., Seehra, J., Somers, W.S., (1999) Cell, 97, pp. 349-360Devedjiev, Y., Popov, A., Atanasov, B., Bartunik, H.D., (1997) J. Mol. Biol., 266, pp. 160-172Dijkstra, B.W., Drenth, J., Kalk, K., Vandermaalen, P.J., (1978) J. Mol. Biol., 124, pp. 53-60Esnouf, R.M., (1997) J. Mol. Graph., 15, pp. 132-134Felsenstein, J., (1989) Cladistics, 5, pp. 164-166Fremont, D.H., Anderson, D.H., Wilson, I.A., Dennis, E.A., Xuong, N.H., (1993) Proc. Natl Acad. Sci. USA, 90, pp. 342-346Frishman, D., Argos, P., (1995) Proteins, 23, pp. 566-579Gerrard, J.M., Robinson, P., Narvey, M., McNicol, A., (1993) Biochem. Cell Biol., 71, pp. 432-439Gutierrez, J.M., Lomonte, B., (1995) Toxicon, 33, pp. 1405-1424Holland, D.R., Clancy, L.L., Muchmore, S.W., Ryde, T.J., Einspahr, H.M., Finzel, B.C., Heinrikson, R.L., Watenpaugh, K.D., (1990) J. Biol. Chem., 265, pp. 17649-17656Holm, L., Sander, C., (1998) Nucleic Acids Res., 26, pp. 316-319Holzer, M., Mackessy, S.P., (1996) Toxicon, 34, pp. 1149-1155Jones, T.A., Zou, J.Y., Cowan, S.W., Kjeldgaard, M., (1991) Acta Cryst., A47, pp. 110-119Keith, C., Feldman, D.S., Deganello, S., Glick, J., Ward, K.B., Jones, E.O., Sigler, P.B., (1981) J. Biol. Chem., 256, pp. 8602-8607Kleywegt, G.J., (1999) Acta Cryst., D55, pp. 1878-1884Kleywegt, G.J., Brünger, A.T., (1996) Structure, 4, pp. 897-904Kleywegt, G.J., Jones, T.A., (1996) Acta Cryst., D52, pp. 829-832Kleywegt, G.J., Jones, T.A., (1998) Acta Cryst., D54, pp. 1119-1131Kraulis, P.J., (1991) J. Appl. Cryst., 24, pp. 946-950Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., (1993) J. Appl. Cryst., 26, pp. 283-291Lawrence, M.C., Colman, P.M., (1993) J. Mol. Biol., 234, pp. 946-950Leahy, D.J., Axel, R., Hendrickson, W.A., (1992) Cell, 68, pp. 1145-1162Lee, W.H., Da Silva Giotto, M.T., Marangoni, S., Toyama, M.H., Polikarpov, I., Garratt, R.C., (2001) Biochemistry, 40, pp. 28-36Lloret, S., Moreno, J.J., (1993) Toxicon, 31, pp. 949-956Lu, G., (2000) J. Appl. Cryst., 33, pp. 176-183Mancuso, L.C., Correa, M.M., Vieira, C.A., Cunha, O.A., Lachat, J.J., De Araújo, H.S., Ownby, C.L., Giglio, J.R., (1995) Toxicon, 33, pp. 615-626Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kezdy, F.J., Heinrikson, R.L., (1984) J. Biol. Chem., 259, pp. 13839-13843Matthews, B.W., (1968) J. Mol. Biol., 33, pp. 491-497Nayal, M., Di Cera, E., (1996) J. Mol. Biol., 256, pp. 228-234Otwinowski, Z., (1993) Proceedings of the CCP4 Study Weekend. Data Collection and Processing, pp. 56-62. , Warrington: Daresbury LaboratoryPan, H., Liu, X.L., Ou-Yang, L.L., Yang, G.Z., Zhou, Y.C., Li, Z.P., Wu, X.F., (1998) Toxicon, 36, pp. 1155-1163Pannu, N.S., Read, R.J., (1996) Acta Cryst., A52, pp. 659-668Perbandt, M., Wilson, J.C., Eschenburg, S., Mancheva, I., Aleksiev, B., Genov, N., Willingmann, P., Betzel, C., (1997) FEBS Lett, 412, pp. 573-577Polikarpov, I., Perles, L.A., De Oliveira, R.T., Oliva, G., Castellano, E.E., Garratt, R., Craievich, A., (1998) J. Synchrotron Rad., 5, pp. 72-76Polikarpov, I., Teplyakov, A., Oliva, G., (1997) Acta Cryst., D53, pp. 734-737Read, R.J., (1986) Acta Cryst., A42, pp. 140-149Renetseder, R., Brunie, S., Dijkstra, B.W., Drenth, J., Sigler, P.B., (1985) J. Biol. Chem., 260, pp. 11627-11634Rice, L.M., Brünger, A.T., (1994) Proteins, 19, pp. 277-290Rychlewski, L., Jaroszewski, L., Li, W., Godzik, A., (2000) Protein Sci., 9, pp. 232-241Sali, A., Blundell, T.L., (1993) J. Mol. Biol., 234, pp. 779-815Schevitz, R.W., (1995) Nature Struct. Biol., 2, pp. 458-465Scott, D.L., Otwinowski, Z., Gelb, M.H., Sigler, P.B., (1990) Science, 250, pp. 1563-1566Scott, D.L., Sigler, P.B., (1994) Adv. Protein Chem., 45, pp. 53-88Shimohigashi, Y., Tani, A., Matsumoto, H., Nakashima, K., Yamaguchi, Y., (1995) J. Biochem., 118, pp. 1037-1044Da Silva Giotto, M.T., Garratt, R.C., Oliva, G., Mascarenhas, Y.P., Giglio, J.R., Cintra, A.C., De Azevedo W.F., Jr., Ward, R.J., (1998) Proteins, 30, pp. 442-454Soares, A.M., Andriao-Escarso, S.H., Bortoleto, R.K., Rodrigues-Simioni, L., Arni, R.K., Ward, R.J., Gutierrez, J.M., Giglio, J.R., (2001) Arch. Biochem. Biophys., 387, pp. 188-196Toyama, M.H., Costa, P.D., Novello, J.C., De Oliveira, B., Giglio, J.R., Da Cruz-Hofling, M.A., Marangoni, S., (1999) J. Protein Chem., 18, pp. 371-378Toyama, M.H., Soares, A.M., Wen-Hwa, L., Polikarpov, I., Giglio, J.R., Marangoni, S., (2000) Biochimie, 82, pp. 245-250Tsai, I.H., Wang, Y.M., Au, L.C., Ko, T.P., Chen, Y.H., Chu, Y.F., (2000) Eur. J. Biochem., 267, pp. 6684-6691Vagin, A., Teplyakov, A., (1997) J. Appl. Cryst., 30, pp. 1022-1025Valdar, W.S., Thornton, J.M., (2001) J. Mol. Biol., 313, pp. 399-416Verity, M.A., (1992) Neurotoxicology, 13, pp. 139-147Ward, R.J., Alves, A.R., Ruggiero Neto, J., Arni, R.K., Casari, G.A., (1998) Protein Eng., 11, pp. 285-29

Encapsulated in silica: genome, proteome and physiology of the thermophilic bacterium Anoxybacillus flavithermus WK1

Sequencing of the complete genome of Anoxybacillus flavithermus reveals enzymes that are required for silica adaptation and biofilm formation

Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

New Structural and Functional Contexts of the Dx[DN]xDG Linear Motif: Insights into Evolution of Calcium-Binding Proteins

Binding of calcium ions (Ca2+) to proteins can have profound effects on their structure and function. Common roles of calcium binding include structure stabilization and regulation of activity. It is known that diverse families – EF-hands being one of at least twelve – use a Dx[DN]xDG linear motif to bind calcium in near-identical fashion. Here, four novel structural contexts for the motif are described. Existing experimental data for one of them, a thermophilic archaeal subtilisin, demonstrate for the first time a role for Dx[DN]xDG-bound calcium in protein folding. An integrin-like embedding of the motif in the blade of a β-propeller fold – here named the calcium blade – is discovered in structures of bacterial and fungal proteins. Furthermore, sensitive database searches suggest a common origin for the calcium blade in β-propeller structures of different sizes and a pan-kingdom distribution of these proteins. Factors favouring the multiple convergent evolution of the motif appear to include its general Asp-richness, the regular spacing of the Asp residues and the fact that change of Asp into Gly and vice versa can occur though a single nucleotide change. Among the known structural contexts for the Dx[DN]xDG motif, only the calcium blade and the EF-hand are currently found intracellularly in large numbers, perhaps because the higher extracellular concentration of Ca2+ allows for easier fixing of newly evolved motifs that have acquired useful functions. The analysis presented here will inform ongoing efforts toward prediction of similar calcium-binding motifs from sequence information alone

ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major

Macroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence

CCP4 Cloud for structure determination and project management in macromolecular crystallography

Nowadays, progress in the determination of three-dimensional macromolecular structures from diffraction images is achieved partly at the cost of increasing data volumes. This is due to the deployment of modern high-speed, high-resolution detectors, the increased complexity and variety of crystallographic software, the use of extensive databases and high-performance computing. This limits what can be accomplished with personal, offline, computing equipment in terms of both productivity and maintainability. There is also an issue of long-term data maintenance and availability of structure-solution projects as the links between experimental observations and the final results deposited in the PDB. In this article, CCP4 Cloud, a new front-end of the CCP4 software suite, is presented which mitigates these effects by providing an online, cloud-based environment for crystallographic computation. CCP4 Cloud was developed for the efficient delivery of computing power, database services and seamless integration with web resources. It provides a rich graphical user interface that allows project sharing and long-term storage for structure-solution projects, and can be linked to data-producing facilities. The system is distributed with the CCP4 software suite version 7.1 and higher, and an online publicly available instance of CCP4 Cloud is provided by CCP4.The following funding is acknowledged: Biotechnology and Biological Sciences Research Council (grant No. BB/L007037/1; grant No. BB/S007040/1; grant No. BB/S007083/1; grant No. BB/S005099/1; grant No. BB/S007105/1; award No. BBF020384/1); Medical Research Council (grant No.MC_UP_A025_1012; grant No. MC_U105184325); Ro¨ntgenA˚ ngstro¨m Cluster (grant No. 349-2013-597); Nederlandse Wetenschappelijke Organisatie (grant No. TKI 16219)

Modular Architecture and Unique Teichoic Acid Recognition Features of Choline-Binding Protein L (CbpL) Contributing to Pneumococcal Pathogenesis

The human pathogen Streptococcus pneumoniae is decorated with a special class of surface-proteins known as choline-binding proteins (CBPs) attached to phosphorylcholine (PCho) moieties from cell-wall teichoic acids. By a combination of X-ray crystallography, NMR, molecular dynamics techniques and in vivo virulence and phagocytosis studies, we provide structural information of choline-binding protein L (CbpL) and demonstrate its impact on pneumococcal pathogenesis and immune evasion. CbpL is a very elongated three-module protein composed of (i) an Excalibur Ca 2+ -binding domain -reported in this work for the very first time-, (ii) an unprecedented anchorage module showing alternate disposition of canonical and non-canonical choline-binding sites that allows vine-like binding of fully-PCho-substituted teichoic acids (with two choline moieties per unit), and (iii) a Ltp-Lipoprotein domain. Our structural and infection assays indicate an important role of the whole multimodular protein allowing both to locate CbpL at specific places on the cell wall and to interact with host components in order to facilitate pneumococcal lung infection and transmigration from nasopharynx to the lungs and blood. CbpL implication in both resistance against killing by phagocytes and pneumococcal pathogenesis further postulate this surface-protein as relevant among the pathogenic arsenal of the pneumococcus.We gratefully acknowledge Karsta Barnekow and Kristine Sievert-Giermann, for technical assistance and Lothar Petruschka for in silico analysis (all Dept. of Genetics, University of Greifswald). We are further grateful to the staff from SLS synchrotron beamline for help in data collection. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG GRK 1870 (to SH) and the Spanish Ministry of Economy and Competitiveness (BFU2014-59389-P to JAH, CTQ2014-52633-P to MB and SAF2012-39760-C02-02 to FG) and S2010/BMD- 2457 (Community of Madrid to JAH and FG).Peer Reviewe

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death