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Virulence-associated protein A (VapA) of Rhodococcus equi is a pathogenicity factor required for the multiplication of virulent R. equi strains within spacious macrophage vacuoles. The production of VapA is characteristic for R. equi isolates from pneumonic foals. VapB and VapN proteins in R. equi isolates from infected pig (VapB) and cattle (VapN) have amino acid sequences very similar to VapA and consequently have been assumed to be its functional correlates. Using model membrane experiments, phagosome pH acidification analysis, lysosome size measurements, protein partitioning, and degradation assays, we provide support for the view that VapA and VapN promote intracellular multiplication of R. equi by neutralizing the pH of the R. equi-containing vacuole. VapB does not neutralize vacuole pH, is not as membrane active as VapA, and does not support intracellular multiplication. This study also shows that the size of the sometimes enormous R. equi-containing vacuoles or the partitioning of purified Vaps into organic phases are not features that have predictive value for virulence of R. equi, whereas the ability of Vaps to increase phagosome pH is coupled to virulence. IMPORTANCE Rhodococcus equi is a major cause of life-threatening pneumonia in foals and occasionally in immunocompromised persons. Virulence-associated protein A (VapA) promotes R. equi multiplication in lung macrophages, which are the major host cells during foal infection. In this study, we compare cellular, biochemical, and biophysical phenotypes associated with VapA to those of VapB (typically produced by isolates from pigs) or VapN (isolates from cattle). Our data support the hypothesis that only some Vaps support multiplication in macrophages by pH neutralization of the phagosomes that R. equi inhabit.

Acta crystallographica / Section D 78(3), 310 - 320 (2022). doi:10.1107/S2059798322000432, InlB, a bacterial agonist of the human receptor tyrosine kinase MET, consists of an N-terminal internalin domain, a central B repeat and three C-terminal GW domains. In all previous structures of full-length InlB or an InlB construct lacking the GW domains (InlB$_{392}$), there was no interpretable electron density for the B repeat. Here, three InlB$_{392}$ crystal structures in which the B repeat is resolved are described. These are the first structures to reveal the relative orientation of the internalin domain and the B repeat. A wild-type structure and two structures of the T332E variant together contain five crystallographically independent molecules. Surprisingly, the threonine-to-glutamate substitution in the B repeat substantially improved the crystallization propensity and crystal quality of the T332E variant. The internalin domain and B repeat are quite rigid internally, but are flexibly linked to each other. The new structures show that inter-domain flexibility is the most likely cause of the missing electron density for the B repeat in previous InlB structures. A potential binding groove between B-repeat strand β2 and an adjacent loop forms an important crystal contact in all five crystallographically independent chains. This region may represent a hydrophobic `sticky patch' that supports protein–protein interactions. This assumption agrees with the previous finding that all known inactivating point mutations in the B repeat lie within strand β2. The groove formed by strand β2 and the adjacent loop may thus represent a functionally important protein–protein interaction site in the B repeat., Published by Wiley, Bognor Regis

We present recent highlights from the most recent operation phases of Wendelstein 7-X, the most advanced stellarator in the world. Stable detachment with good particle exhaust, low impurity content, and energy confinement times exceeding 100 ms, have been maintained for tens of seconds. Pellet fueling allows for plasma phases with reduced ion-temperature-gradient turbulence, and during such phases, the overall confinement is so good (energy confinement times often exceeding 200 ms) that the attained density and temperature profiles would not have been possible in less optimized devices, since they would have had neoclassical transport losses exceeding the heating applied in W7-X. This provides proof that the reduction of neoclassical transport through magnetic field optimization is successful. W7-X plasmas generally show good impurity screening and high plasma purity, but there is evidence of longer impurity confinement times during turbulence-suppressed phases. This work has been carried out within the framework of the EUROfusion Consortium and has received funding from the Euratom research and training programme 2014-2018 and 2019-2020 under Grant Agreement No. 633053. Peer Reviewed Article signat per 497 autors/es: Thomas Sunn Pedersen1,2,∗ , I. Abramovic3, P. Agostinetti4, M. Agredano Torres1, S. Äkäslompolo1, J. Alcuson Belloso1, P. Aleynikov1, K. Aleynikova1, M. Alhashimi1, A. Ali1, N. Allen5, A. Alonso6, G. Anda7, T. Andreeva1, C. Angioni8, A. Arkhipov8, A. Arnold1, W. Asad8, E. Ascasibar6, M.-H. Aumeunier9, K. Avramidis10, E. Aymerich11, S.-G. Baek3, J. Bähner1, A. Baillod12, M. Balden1, M. Balden8, J. Baldzuhn1, S. Ballinger3, M. Banduch1, S. Bannmann1, A. Banon Navarro8, A. Bañon Navarro ´ 1, T. Barbui13, C. Beidler1, C. Belafdil9, A. Bencze7, A. Benndorf1, M. Beurskens1, C. Biedermann1, O. Biletskyi14, B. Blackwell15, M. Blatzheim1, T. Bluhm1, D. Böckenhoff1, G. Bongiovi16, M. Borchardt1, D. Borodin17, J. Boscary8, H. Bosch1,18, T. Bosmann19, B. Böswirth8, L. Böttger1, A. Bottino8, S. Bozhenkov1, R. Brakel1, C. Brandt1, T. Bräuer1, H. Braune1, S. Brezinsek17, K. Brunner1, S. Buller1, R. Burhenn1, R. Bussiahn1, B. Buttenschön1, A. Buzás7, V. Bykov1, I. Calvo6, K. Camacho Mata1, I. Caminal20, B. Cannas11, A. Cappa6, A. Carls1, F. Carovani1, M. Carr21, D. Carralero6, B. Carvalho22, J. Casas20, D. Castano-Bardawil17, F. Castejon6, N. Chaudhary1, I. Chelis23, A. Chomiczewska24, J.W. Coenen13,17, M. Cole1, F. Cordella25, Y. Corre9, K. Crombe26, G. Cseh7, B. Csillag7, H. Damm1, C. Day10, M. de Baar27, E. De la Cal6, S. Degenkolbe1, A. Demby13, S. Denk3, C. Dhard1, A. Di Siena8,28, A. Dinklage12, T. Dittmar17, M. Dreval14, M. Drevlak1, P. Drewelow1, P. Drews17, D. Dunai7, E. Edlund3, F. Effenberg29, G. Ehrke1, M. Endler1, D.A. Ennis5, F.J. Escoto6, T. Estrada6, E. Fable8, N. Fahrenkamp1, A. Fanni11, J. Faustin1, J. Fellinger1, Y. Feng1, W. Figacz4, E. Flom13, O. Ford1, T. Fornal24, H. Frerichs13, S. Freundt1, G. Fuchert1, M. Fukuyama30, F. Füllenbach1, G. Gantenbein10, Y. Gao1, K. Garcia13, J.M. García Regaña6, I. García-Cortés6, J. Gaspar31, D.A. Gates29, J. Geiger1, B. Geiger13, L. Giudicotti32, A. González6, A. Goriaev26,33, D. Gradic1, M. Grahl1, J.P. Graves12, J. Green13, E. Grelier9, H. Greuner8, S. Groß1, H. Grote1, M. Groth34, M. Gruca24, O. Grulke1,35, M. Grün1, J. Guerrero Arnaiz1, S. Günter8, V. Haak1, M. Haas1, P. Hacker1, A. Hakola36, A. Hallenbert1, K. Hammond29, X. Han17,37, S.K. Hansen3, J.H. Harris38, H. Hartfuß1, D. Hartmann1, D. Hathiramani1, R. Hatzky8, J. Hawke39, S. Hegedus7, B. Hein8, B. Heinemann8, P. Helander12, S. Henneberg1, U. Hergenhahn8,40, C. Hidalgo6, F. Hindenlang8, M. Hirsch1, U. Höfel1, K.P. Hollfeld17, A. Holtz1, D. Hopf8, D. Höschen17, M. Houry9, J. Howard19, X. Huang41, M. Hubeny17, S. Hudson29, K. Ida9, Y. Igitkhanov10, V. Igochine8, S. Illy10, C. Ionita-Schrittwieser42, M. Isobe39, M. Jabłczynska ´ 24, S. Jablonski24, B. Jagielski1, M. Jakubowski1, A. Jansen van Vuuren1, J. Jelonnek10, F. Jenko8, F. Jenko8, T. Jensen35, H. Jenzsch1, P. Junghanns8, J. Kaczmarczyk24, J. Kallmeyer1, U. Kamionka1, M. Kandler8, S. Kasilov43, Y. Kazakov26, D. Kennedy1, A. Kharwandikar1, M. Khokhlov1, C. Kiefer8, C. Killer1, A. Kirschner17, R. Kleiber1, T. Klinger12, S. Klose1, J. Knauer1, A. Knieps17, F. Köchl44, G. Kocsis7, Ya.I. Kolesnichenko45, A. Könies1, R. König1, J. Kontula34, P. Kornejew1, J. Koschinsky, M.M. Kozulia14, A. Krämer-Flecken17, R. Krampitz1, M. Krause1, N. Krawczyk24, T. Kremeyer1, L. Krier10, D.M. Kriete5, M. Krychowiak1, I. Ksiazek46, M. Kubkowska24, M. Kuczynski1, G. Kühner1, A. Kumar15, T. Kurki-Suonio34, S. Kwak1, M. Landreman47, P.T. Lang8, A. Langenberg1, H.P. Laqua12, H. Laqua1, R. Laube1, S. Lazerson1, M. Lewerentz1, C. Li17, Y. Liang17, Ch. Linsmeier17, J. Lion1, A. Litnovsky17,48, S. Liu37, J. Lobsien1, J. Loizu12, J. Lore38, A. Lorenz1, U. Losada6, F. Louche26, R. Lunsford29, V. Lutsenko45, M. Machielsen12, F. Mackel8, J. Maisano-Brown3, O. Maj8, D. Makowski49, G. Manduchi50, E. Maragkoudakis6, O. Marchuk17, S. Marsen1, E. Martines4, J. Martinez-Fernandez6, M. Marushchenko1, S. Masuzaki41, D. Maurer5, M. Mayer8, K.J. McCarthy6, O. Mccormack4, P. McNeely1, H. Meister8, B. Mendelevitch8, S. Mendes1, A. Merlo1, A. Messian26, A. Mielczarek49, O. Mishchenko1, B. Missal1, R. Mitteau9, V.E. Moiseenko14, A. Mollen1, V. Moncada9, T. Mönnich1, T. Morisaki41, D. Moseev1, G. Motojima41, S. Mulas6, M. Mulsow1, M. Nagel1, D. Naujoks1, V. Naulin35, T. Neelis19, H. Neilson29, R. Neu8, O. Neubauer17, U. Neuner1, D. Nicolai17, S.K. Nielsen35, H. Niemann1, T. Nishiza1, T. Nishizawa1, T. Nishizawa8, C. Nührenberg1, R. Ochoukov8, J. Oelmann17, G. Offermanns17 K. Ogawa41, S. Okamura41, J. Ölmanns17, J. Ongena26, J. Oosterbeek1, M. Otte1, N. Pablant29, N. Panadero Alvarez6, N. Panadero Alvarez6, A. Pandey1, E. Pasch1, R. Pavlichenko14, A. Pavone1, E. Pawelec46, G. Pechstein1, G. Pelka24, V. Perseo1, B. Peterson41, D. Pilopp1, S. Pingel1, F. Pisano11, B. Plöckl8, G. Plunk1, P. Pölöskei1, B. Pompe2, A. Popov51, M. Porkolab3, J. Proll19, M.J. Pueschel19,27, M.-E. Puiatti52, A. Puig Sitjes1, F. Purps1, K. Rahbarnia1, M. Rasinski ´ 17, J. Rasmussen35, A. Reiman29, F. Reimold1, M. Reisner8, D. Reiter17, M. Richou9, R. Riedl8, J. Riemann1, K. Riße1, G. Roberg-Clark1, V. Rohde8, J. Romazanov17, D. Rondeshagen1, P. Rong1, L. Rudischhauser1, T. Rummel1, K. Rummel1, A. Runov1, N. Rust1, L. Ryc24, P. Salembier20, M. Salewski35, E. Sanchez6, S. Satake41, G. Satheeswaran17, J. Schacht1, E. Scharff1, F. Schauer8, J. Schilling1, G. Schlisio1, K. Schmid8, J. Schmitt5, O. Schmitz13, W. Schneider1, M. Schneider1, P. Schneider8, R. Schrittwieser42, T. Schröder1, M. Schröder1, R. Schroeder1, B. Schweer26, D. Schwörer1, E. Scott1, E. Scott8, B. Shanahan1, G. Sias11, P. Sichta29, M. Singer1, P. Sinha29, S. Sipliä34, C. Slaby1, M. Sleczka53, H. Smith1, J. Smoniewski54, E. Sonnendrücker8, M. Spolaore4, A. Spring1, R. Stadler8, D. Stanczak24, T. Stange1, I. Stepanov26, L. Stephey13, J. Stober8, U. Stroth8,55, E. Strumberger8, C. Suzuki41, Y. Suzuki41, J. Svensson1, T. Szabolics7, T. Szepesi7, M. Szücs7, F.L. Tabares6, N. Tamura41, A. Tancetti35, C. Tantos10, J. Terry3, H. Thienpondt6, H. Thomsen1, M. Thumm10, J.M. Travere9, P. Traverso5, J. Tretter8, E. Trier8, H. Trimino Mora1, T. Tsujimura41, Y. Turkin1, A. Tykhyi45, B. Unterberg17, P. van Eeten1, B.Ph. van Milligen6, M. van Schoor26, L. Vano1, S. Varoutis10, M. Vecsei7, L. Vela56, J.L. Velasco6, M. Vervier17, N. Vianello50, H. Viebke1, R. Vilbrandt1, G. Vogel8, N. Vogt1, C. Volkhausen1, A. von Stechow1, F. Wagner1, E. Wang17, H. Wang57, F. Warmer1, T. Wauters26, L. Wegener1, T. Wegner1, G. Weir1, U. Wenzel1, A. White3, F. Wilde1, F. Wilms1, T. Windisch1, M. Winkler1, A. Winter1, V. Winters1, R. Wolf118, A.M. Wright29, G.A. Wurden39, P. Xanthopoulos1, S. Xu17, H. Yamada58, H. Yamaguchi41, M. Yokoyama41, M. Yoshinuma41, Q. Yu8, M. Zamanov14, M. Zanini1, M. Zarnstorff29, D. Zhang1, S. Zhou17, J. Zhu1, C. Zhu29, M. Zilker8, A. Zocco1, H. Zohm8, S. Zoletnik7 and L. Zsuga7 // 1 Max Planck Institute for Plasma Physics, Garching and Greifswald, Germany: 2 University of Greifswald, Greifswald, Germany; 3 Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, United States of America; 4 Consorzio RFX, Corso Stati Uniti, 4-35127 Padova, Italy; 5 Auburn University, Auburn, AL 36849, United States of America; 6 CIEMAT, Avenida Complutense, 40, 28040 Madrid, Spain; 7 Center for Energy Research, Konkoly-Thegeut 29-33, 1121 Budapest, Hungary; 8 Max-Planck-Institute for Plasma Physics, Boltzmannstraße 2, 85748 Garching bei München, Germany; 9 CEA Cadarache, 13115 Saint-Paul-lez-Durance, France; 10 Karlsruhe Institute of Technology, Kaiserstr. 12, 76131 Karlsruhe, Germany; 11 University of Cagliari, Via Universita, 40, 09124 Cagliari, Italy; 12 École Polytechnique Fédérale de Lausanne, Swiss Plasma Center, CH-1015 Lausanne, Switzerland; 13 University of Wisconsin–Madison, Engineering Drive, Madison, WI 53706, United States of America; 14 Institute of Plasma Physics, National Science Center ‘Kharkiv Institute of Physics and Technology’, Kharkiv, Ukraine; 15 The Australian National University, Acton ACT 2601, Canberra, Australia; 16 Department of Engineering, University of Palermo, Viale delle Scienze, Edificio 6, Palermo, 90128, Italy; 17 Forschungszentrum Jülich GmbH, Institut für Energie-und Klimaforschung—Plasmaphysik, 52425 Jülich, Germany; 18 Technical University of Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany; 19 Eindhoven University of Technology, 5600 MB Eindhoven, Netherlands; 20 Universitat Politècnica de Catalunya. BarcelonaTech, C. Jordi Girona, 31, 08034 Barcelona, Spain; 21 Culham Center for Fusion Energy, Abingdon OX14 3EB, United Kingdom; 22 Instituto de Plasmas e Fusao Nuclear, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; 23 Department of Physics, National and Kapodistrian University of Athens, 15784 Athens, Greece; 24 Institute of Plasma Physics and Laser Microfusion, 23 Hery Str., 01-497 Warsaw, Poland; 25 ENEA—Centro Ricerche Frascati, Via Enrico Fermi, 45, 00044 Frascati RM, Italy; 26 Laboratory for Plasma Physics, LPP-ERM/KMS, TEC Partner, B-1000 Brussels, Belgium; 27 Dutch Institute for Fundamental Energy Research, PO Box 6336, 5600 HH Eindhoven, Netherlands; 28 University of Texas, Austin, TX, United States of America; 29 Princeton Plasma Physics Laboratory, Princeton, NJ 08543, United States of America; 30 Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395, Japan; 31 Aix-Marseille University, Jardin du Pharo, 58 Boulevard Charles Livon, 13007, Marseille, France; 32 Department of Physics and Astronomy, Padova University, Via Marzolo 8, 35131 Padova, Italy; 33 Department of Applied Physics, Ghent University, Sint-Pietersnieuwstraat 41 B4, 9000 Ghent, Belgium; 34 Aalto University, 02150 Espoo, Finland; 35 Department of Physics, Technical University of Denmark, Anker Engelunds Vej, 2800 Kgs Lyngby, Denmark; 36 VTT Technical Research Center of Finland Ltd., PO Box 1000, FI-02044 VTT, Finland; 37 Institute of Plasma Physics, Chinese Academy of Sciences, 230031 Hefei, Anhui, China; 38 Oak Ridge National Laboratory, 1 Bethel Valley Rd, Oak Ridge, TN 37830, United States of America; 39 Los Alamos National Laboratory, NM 87545, United States of America; 40 Fritz-Haber-Institut der Max-Planck-Gesellschaft, 14195 Berlin, Germany; 41 National Institute for Fusion Science, National Institutes of Natural Sciences, 322-6 Oroshi-cho, Toki, Gifu Prefecture 509-5292, Japan; 42 Institute for Ion Physics and Applied Physics, University of Innsbruck, Innsbruck, Austria; 43 Graz University of Technology, Rechbauerstraße 12, 8010 GRAZ, Austria; 44 Austrian Academy of Science, Doktor-Ignaz-Seipel-Platz 2, 1010 Wien, Austria; 45 Institute for Nuclear Research, prospekt Nauky 47, Kyiv 03028, Ukraine; 46 University of Opole, plac Kopernika 11a, 45-001 Opole, Poland; 47 University of Maryland, Paint Branch Drive, College Park, MA 20742, United States of America; 48 National Research Nuclear University MEPhI, 115409 Moscow, Russian Federation; 49 Department of Microelectronics and Computer Science, Lodz University of Technology, Wolczanska 221/223, 90-924 Lodz, Poland; 50 Consiglio Nazionale delle Ricerche, Piazzale Aldo Moro, 7, 00185 Roma, Italy; 51 Ioffe Physical-Technical Institute of the Russian Academy of Sciences, 26 Politekhnicheskaya, St Petersburg 194021, Russian Federation; 52 Istituto di Fisica del Plasma Piero Caldirola, Via Roberto Cozzi, 53, 20125 Milano, Italy; 53 University of Szczecin, 70-453, aleja Papieza Jana Pawła II 22A, Szczecin, Poland; 54 Lawrence University, 711 E Boldt Way, Appleton, WI 54911, United States of America; 55 Physik-Department E28, Technische Universität München, 85747 Garching, Germany; 56 Universidad Carlos III de Madrid, Av. de la Universidad, 30 Madrid, Spain; 57 Yale University, New Haven, CT 06520, United States of America; 58 University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chhiab 277-0882, Japan Objectius de Desenvolupament Sostenible::7 - Energia Assequible i No Contaminant Objectius de Desenvolupament Sostenible::7 - Energia Assequible i No Contaminant::7.a - Per a 2030, augmentar la cooperació internacional per tal de facilitar l’accés a la investigació i a les tecnolo­gies energètiques no contaminants, incloses les fonts d’energia renovables, l’eficiència energètica i les tecnologies de combustibles fòssils avançades i menys contaminants, i promoure la inversió en infraestructures energètiques i tecnologies d’energia no contaminant

To facilitate the estimation of heat loads on plasma-facing components in fusion devices in various different magnetic geometries, a heat load proxy model was developed based on anisotropic diffusion. In this work, this model is compared to the so-called field-line diffusion approach. To facilitate the evaluation of these models, a novel synthetic camera-based approach for obtaining heat load distributions from Monte Carlo samples was also developed and implemented. With the assistance of this synthetic camera, heat load predictions for the Wendelstein 7-X divertor were obtained and compared with infrared camera observations. It was found that the anisotropic diffusion-based model achieved a closer match to infrared camera observations, while still being suitable in computational effort for large magnetic configuration database scans.

Wendelstein 7-X (W7-X), the largest advanced stellarator, is built to demonstrate high power, high performance quasi-continuous operation. Therefore, in the recent campaign, experiments were performed to prepare for long pulse operation, addressing three critical issues: the development of stable detachment, control of the heat and particle exhaust, and the impact of leading edges on plasma performance. The heat and particle exhaust in W7-X is realized with the help of an island divertor, which utilizes large magnetic islands at the plasma boundary. This concept shows very efficient heat flux spreading and favourable scaling with input power. Experiments performed to overload leading edges showed that the island divertor yields good impurity screening. A highlight of the recent campaign was a robust detachment scenario, which allowed reducing power loads even by a factor of ten. At the same time, neutral pressures at the pumping gap entrance yielded the particle removal rate close to the values required for stable density control in steady-state operation.

Acta crystallographica / F 77(8), 246 - 253 (2021). doi:10.1107/S2053230X2100738X, Virulence-associated proteins (Vaps) contribute to the virulence of the pathogen Rhodococcus equi, but their mode of action has remained elusive. All Vaps share a conserved core of about 105 amino acids that folds into a compact eight-stranded antiparallel β-barrel with a unique topology. At the top of the barrel, four loops connect the eight β-strands. Previous Vap structures did not show concave surfaces that might serve as a ligand-binding site. Here, the structure of VapB in a new crystal form was determined at 1.71 Å resolution. The asymmetric unit contains two molecules. In one of them, the loop regions at the top of the barrel adopt a different conformation from other Vap structures. An outward movement of the loops results in the formation of a hydrophobic cavity that might act as a ligand-binding site. This lends further support to the hypothesis that the structural similarity between Vaps and avidins suggests a potential binding function for Vaps., Published by Blackwell, Oxford [u.a.]

Xcc4156 is a flavin-dependent halogenase from Xanthomonas campestris. Here, an apo structure is presented which, in combination with soaking experiments, provides functional insight into the binding of FAD and bromide to the halogenase., Flavin-dependent halogenases regioselectively introduce halide substituents into electron-rich substrates under mild reaction conditions. For the enzyme Xcc4156 from Xanthomonas campestris, the structure of a complex with the cofactor flavin adenine dinucleotide (FAD) and a bromide ion would be of particular interest as this enzyme exclusively brominates model substrates in vitro. Apo Xcc4156 crystals diffracted to 1.6 Å resolution. The structure revealed an open substrate-binding site lacking the loop regions that close off the active site and contribute to substrate binding in tryptophan halogenases. Therefore, Xcc4156 might accept larger substrates, possibly even peptides. Soaking of apo Xcc4156 crystals with FAD led to crumbling of the intergrown crystals. Around half of the crystals soaked with FAD did not diffract, while in the others there was no electron density for FAD. The FAD-binding loop, which changes its conformation between the apo and the FAD-bound form in related enzymes, is involved in a crystal contact in the apo Xcc4156 crystals. The conformational change that is predicted to occur upon FAD binding would disrupt this crystal contact, providing a likely explanation for the destruction of the apo crystals in the presence of FAD. Soaking with only bromide did not result in bromide bound to the catalytic halide-binding site. Simultaneous soaking with FAD and bromide damaged the crystals more severely than soaking with only FAD. Together, these latter two observations suggest that FAD and bromide bind to Xcc4156 with positive cooperativity. Thus, apo Xcc4156 crystals provide functional insight into FAD and bromide binding, even though neither the cofactor nor the halide is visible in the structure.

Flavin-dependent halogenases chlorinate or brominate their substrates in an environmentally friendly manner, only requiring the cofactor reduced flavin adenine dinucleotide (FADH2), oxygen, and halide salts. The tryptophan 6-halogenase Thal exhibits two flexible loops, which become ordered (substrate-binding loop) or adopt a closed conformation (FAD loop) upon substrate or cofactor binding. Here, we describe the structure of NHis-Thal-RebH5 containing an N-terminal His-tag from pET28a, which crystallized in a different space group (P21) and, surprisingly, diffracted to a higher resolution of 1.63 &Aring, than previously deposited Thal structures (P64, ~2.2 &Aring, ) with cleaved His-tag. Interestingly, the binding of glycine in the active site can induce an ordered conformation of the substrate-binding loop.

Protein science 28(12), 2112-2118 (2019). doi:10.1002/pro.3739, Flavin-dependent halogenases require reduced flavin adenine dinucleotide (FADH2 ), O2 , and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6-halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH2 by flavin reductases., Published by Protein Society, Bethesda, Md.

JBC papers in press 294(7), 2529 - 2542 (2019). doi:10.1074/jbc.RA118.005393, Flavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH2, oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate trypto-phan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 Å resolution. The structure revealed that the C6 of tryptophan is positioned next to the ϵ-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of l-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases., Published by American Soc. for Biochemistry and Molecular Biology, Bethesda, MD.

Fluorescence methods are important tools in modern biology. Direct labeling of biomolecules with a fluorophore might, however, change interaction surfaces. Here, we introduce a competitive binding assay in combination with fluorescence correlation spectroscopy that reports binding affinities of both labeled and unlabeled biomolecules to their binding target. We investigated how fluorophore labels at different positions of a DNA oligonucleotide affect hybridization to a complementary oligonucleotide and found dissociation constants varying within 2 orders of magnitude. We next demonstrated that placing a fluorophore label at position Leu280 in the protein ligand internalin B does not alter the binding affinity to the MET receptor tyrosine kinase, compared to unlabeled internalin B. Our approach is simple to implement and can be applied to investigate the influence of fluorophore labels in a large variety of biomolecular interactions.

CagL is an essential pilus surface component of the virulence-associated type IV secretion system (T4SS) employed by Helicobacterpylori to translocate the oncogenic effector protein CagA into human gastric epithelial cells. CagL contains an RGD motif and integrin alpha5 beta1 is widely accepted as its host cell receptor. Here, we show that CagL binds integrin alphaV beta6 with substantially higher affinity and that this interaction is functionally important. Cell surface expression of alphaV beta6 on various cell lines correlated perfectly with cell adhesion to immobilized CagL and with binding of soluble CagL to cells. We found no such correlation for alpha5 beta1 . The purified alphaV beta6 ectodomain bound CagL with high affinity. This interaction was highly specific, as the affinity of CagL for other RGD-binding integrins was two to three orders of magnitude weaker. Mutation of either conserved leucine in the CagL RGDLXXL motif, a motif that generally confers specificity for integrin alphaV beta6 and alphaV beta8 , lowered the affinity of CagL for alphaV beta6 . Stable expression of alphaV beta6 in alphaV beta6 -negative but alpha5 beta1 -expressing human cells promoted two hallmarks of the functional H.pylori T4SS, namely translocation of CagA into host cells and induction of interleukin-8 secretion by host cells. These findings suggest that integrin alphaV beta6 , although not essential for T4SS function, represents an important host cell receptor for CagL. © 2019 Federation of European Biochemical Societies.

Flavin-dependent halogenases can be used as biocatalysts because they regioselectively halogenate their substrates under mild reaction conditions. New halogenases with novel substrate specificities will add to the toolbox of enzymes available to organic chemists. HalX, the product of the xcc-b100_4193 gene, is a putative flavin-dependent halogenase from Xanthomonas campestris. The enzyme was recombinantly expressed and crystallized in order to aid in identifying its hitherto unknown substrate. Native data collected to a resolution of 2.5 Å showed indications of merohedral twinning in a hexagonal lattice. Attempts to solve the phase problem by molecular replacement failed. Here, a detailed analysis of the suspected twinning is presented. It is most likely that the crystals are trigonal (point group 3) and exhibit perfect hemihedral twinning so that they appear to be hexagonal (point group 6). As there are several molecules in the asymmetric unit, noncrystallographic symmetry may complicate twinning analysis and structure determination.

The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto-oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single-particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B-bound MET. We show that internalin B-bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B-bound receptors than for resting MET. Results of single-particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.

SummaryBackground 8-Methoxypsoralen–UVA (PUVA) and narrowband ultraviolet B (NB-UVB) are well-established treatments for chronic plaque-type psoriasis vulgaris. However, long-term risks of PUVA therapy include premature skin ageing and squamous cell carcinoma. Objectives To develop a device for targeted UV therapy of psoriatic plaques with protection of the healthy adjacent skin to reduce the risk for premature skin ageing and squamous cell carcinoma. Methods In total 28 patients with exacerbated psoriasis vulgaris were treated with the digital phototherapy device skintrek® [cream PUVA (n = 8), bath PUVA (n = 11) and UVB (n = 9)] or with conventional bath PUVA (n = 9) or NB-UVB (n = 4). Results The local Psoriasis Area and Severity Index (PASI) score was significantly reduced from a mean of 6·25 at baseline to 2·75 at the end of therapy in the skintrek cream PUVA group, from 6·4 to 3·0 in the skintrek bath PUVA group and from 5·5 to 2·0 in the skintrek UVB group. Treatment with skintrek cream PUVA reduced the mean local PASI by 54%, while skintrek bath PUVA did so by 51% and skintrek UVB by 63%. Targeted skintrek PUVA and skintrek UVB of inflamed psoriatic skin avoided skin pigmentation and were not inferior to conventional bath PUVA and NB-UVB therapy regimens. Conclusions Targeted UV therapy of psoriatic plaques with the digital phototherapy device skintrek is as effective as conventional cream and bath PUVA, as well as NB-UVB, while simultaneously sparing the healthy adjacent skin. It therefore potentially reduces the carcinogenic risk, reduces premature skin ageing and avoids tanning of healthy surrounding skin.

A constitutively dimeric truncated variant of internalin B (InlB321-CD), acting as stimulator of the receptor tyrosine kinase MET, was tested for dermal wound-healing potential. Due to a lack of the endogenous MET agonist HGF/SF in chronic wounds, HGF/SF substitution by an InlB321-CD-loaded hydrogel might be beneficial in chronic wound therapy. In this study, InlB321-CD in solution and incorporated in a hydrogel was tested for mitogenic effects on immortalized human dermal keratinocytes (HaCaT) with an MTT assay. Cell migration was investigated with a scratch assay on primary keratinocytes (PHK) and on HaCaT. For the latter, scratching needed to be mitomycin C-controlled. InlB321-CD effects on a model of human skin were analyzed histologically with respect to viability. InlB321-CD led to dose-dependent proliferative effects on HaCaT cells whereas the equimolar dose of monomeric InlB321 did not. Upon hydrogel incorporation of InlB321-CD its mitogenic activity for HaCaT cells was maintained thus confirming the hydrogel as a promising drug delivery system. Motogenic effects were shown on both HaCaT and PHK cells. InlB321-CD neither possesses cytotoxic effects on the viability of a human skin model nor alters its organotypic cell morphology.

A new crystal form of theHelicobacter pyloritype IV secretion system (T4SS) pilus protein CagL is described here. In contrast to two previously reported monomeric structures, CagL forms a three-dimensional domain-swapped dimer. CagL dimers can arise during refolding from inclusion bodies or can form spontaneously from purified monomeric CagL in the crystallization conditions. Monomeric CagL forms a three-helix bundle, with which the N-terminal helix is only loosely associated. In the new crystal form, the N-terminal helix is missing. The domain swap is owing to exchange of the C-terminal helix between the two protomers of a dimer. A loop-to-helix transition results in a long helix of 108 amino acids comprising the penultimate and the last helix of the monomer. The RGD motif of dimeric CagL adopts an α-helical conformation. In contrast to the previously reported structures, the conserved and functionally important C-terminal hexapeptide is resolved. It extends beyond the three-helix bundle as an exposed helical appendage. This new crystal form contributes to the molecular understanding of CagL by highlighting rigid and flexible regions in the protein and by providing the first view of the C-terminus. Based on the structural features, a previously unrecognized homology between CagL and CagI is discussed.

Background The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. Results To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Conclusions Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.

The structural basis of ligand-induced dimerization of the receptor tyrosine kinase MET by its natural ligand hepatocyte growth factor/scatter factor (HGF/SF) is not well understood. However, interesting insight into the molecular mechanism of MET dimerization has emerged from crystal structures of MET in complex with a bacterial agonist, the invasion protein internalin B (InlB) from pathogenic Listeria monocytogenes. MET activation by InlB promotes uptake of bacteria into host cells. Structural and biophysical data suggest that InlB is monomeric on its own but dimerizes upon binding to the membrane-anchored MET receptor promoting the formation of a signaling active 2:2 complex. The dimerization interface is small and unusually located on the convex side of the curved InlB leucine-rich repeat (LRR) domain. As InlB does not dimerize in solution, the dimerization site could only be identified by studying packing contacts of InlB in various crystal forms and had to be proven by scrutinizing its biological relevance in cellular assays. InlB dimerization is thus an example of a low-affinity contact that appears irrelevant in solution but becomes physiologically significant in the context of 2-dimensional diffusion restricted to the membrane plane. The resulting 2:2 InlB: MET complex has an InlB dimer at its center with one MET molecule bound peripherally to each InlB. This model of ligand-mediated MET dimerization may serve as a blue-print to understand MET activation by NK1, a naturally occurring HGF/SF splice variant and MET agonist. Crystal structures of NK1 repeatedly show a NK1 dimer, in which residues implicated in MET-binding are located on the outside. Thus, MET dimerization by NK1 may also be ligand-mediated with a NK1 dimer at the center of the 2:2 complex with one MET molecule bound peripherally to each NK1. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases. (C) 2012 Elsevier B.V. All rights reserved.

The yeast peroxisomal hydrolase Lpx1 belongs to the alpha/beta-hydrolase superfamily. In the absence of Lpx1, yeast peroxisomes show an aberrant vacuolated morphology similar to what is found in peroxisomal disorder patients. Here, we present the crystal structure of Lpx1 determined at a resolution of 1.9A. The structure reveals the complete catalytic triad with an unusual location of the acid residue after strand beta6 of the canonical alpha/beta-hydrolase fold. A four-helix cap domain covers the active site. The interface between the alpha/beta-hydrolase core and the cap domain forms the potential substrate binding site, which may also comprise the tunnel that leads into the protein interior and widens into a cavity. Two further tunnels connect the active site to the protein surface, potentially facilitating substrate access. Lpx1 is a homodimer. The alpha/beta-hydrolase core folds of the two protomers form the dimer contact site. Further dimerization contacts arise from the mutual embracement of the cap domain of one protomer by the non-canonical C-terminal helix of the other, resulting in a total buried surface area of some 6000A(2). The unusual C-terminal helix sticks out from the core fold to which it is connected by an extended flexible loop. We analyzed whether this helix is required for dimerization and for import of the dimer into peroxisomes using biochemical assays in vitro and a microscopy-based interaction assay in mammalian cells. Surprisingly, the C-terminal helix is dispensable for dimerization and dimer import. The unusually robust self-interaction suggests that Lpx1 is imported into peroxisomes as dimer. Copyright 2011 Elsevier Inc. All rights reserved.

Host cell invasion by the facultative intracellular pathogen Listeria monocytogenes requires the invasion protein InlB in many cell types. InlB consists of an N-terminal internalin domain that binds the host cell receptor tyrosine kinase Met and C-terminal GW domains that bind to glycosaminoglycans (GAGs). Met binding and activation is required for host cell invasion, while the interaction between GW domains and GAGs enhances this effect. Soluble InlB elicits the same cellular phenotypes as the natural Met ligand hepatocyte growth factor/scatter factor (HGF/SF), e.g. cell scatter. So far, little is known about the central part of InlB, the B-repeat. Here we present a structural and functional characterization of the InlB B-repeat. The crystal structure reveals a variation of the β-grasp fold that is most similar to small ubiquitin-like modifiers (SUMOs). However, structural similarity also suggests a potential evolutionary relation to bacterial mucin-binding proteins. The B-repeat defines the prototype structure of a hitherto uncharacterized domain present in over a thousand bacterial proteins. Generally, this domain probably acts as a spacer or a receptor-binding domain in extracellular multi-domain proteins. In cellular assays the B-repeat acts synergistically with the internalin domain conferring to it the ability to stimulate cell motility. Thus, the B-repeat probably binds a further host cell receptor and thereby enhances signaling downstream of Met.

Listeria monocytogenes, a Gram-positive bacterium, is the causative agent for the disease called listeriosis. This pathogen utilizes host cell surface proteins such as E-cadherin or c-Met in order to invade eukaryotic cells. The invasion via c-Met depends on the bacterial protein InlB that activates c-Met phosphorylation and internalization mimicking in many regards HGF, the authentic c-Met ligand. In this paper, we demonstrate that the activation of c-Met induced by InlB is dependent on CD44v6, a member of the CD44 family of transmembrane glycoproteins. Inhibiting CD44v6 by means of a blocking peptide, a CD44v6 antibody or CD44v6-specific siRNA prevents the activation of c-Met induced by InlB. Subsequently, signalling, scattering and the entry of InlB-coated beads into host cells are also impaired by CD44v6 blocking reagents. For the entry process, ezrin, a protein that links the CD44v6 cytoplasmic domain to the cytoskeleton, is required as well. Most importantly, this collaboration between c-Met and CD44v6 contributes to the invasion of L. monocytogenes into target cells as demonstrated by a drastic decrease in bacterial invasion in the presence of blocking agents such as the CD44v6 peptide or antibody.

International audience; BACKGROUND: Animal studies have demonstrated better embryo development in vivo than in vitro. This pilot study tested the feasibility of using a novel in utero culture system (IUCS) to obtain normal human fertilization and embryo development. METHODS: The IUCS device comprised a perforated silicone hollow tube. The study included 13 patients (

Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met.

We present a fusion system suited to determine the crystal structure of small disulfide-rich proteins. McoEeTI, a hybrid inhibitor cystine knot microprotein, was produced as a soluble fusion to a catalytically inactive variant of the RNAse barnase in Escherichia coli. Functioning as a versatile tag, barnase facilitated purification, crystallization and high-resolution structure determination. Flexibility of the linker region allows for different relative orientations of barnase and the fusion partner in two crystallographically independent molecules and may thereby facilitate crystal packing. Nevertheless, the linker region is well ordered in both molecules. This system may prove more generally useful to determine the crystal structure of peptides and small proteins.

Adhesion and invasion of pathogenic bacteria represent the important initial step of infection. Pathogens utilize surface-located adhesins/invasins for specific interaction with host cell receptors. The three-dimensional structures of a number of adhesins/invasins show that many are elongated molecules containing domains commonly found in eukaryotic proteins. Similar folds are employed repeatedly to target different receptors.

Editor—Endometrial cancer (EC) is the second most common malignancy in the hereditary non-polyposis colorectal cancer (HNPCC) syndrome.1 In a recent large study, cumulative cancer incidences by the age of 70 in HNPCC mutation carriers were: colorectal 82%, endometrial 60%, gastric 13%, and ovarian 12%.2 Interestingly, in female mutation carriers the incidence of endometrial cancer (60%) exceeded that of colorectal cancer (CRC) (54%), as had been suggested earlier.2 3 Predisposition to HNPCC is the result of germline mutations in the mismatch repair genes.4 Detectable mutations in the two major genes, MLH1 and MSH2 , account for some 3% of all colorectal cancers.5 One might therefore assume that a similar proportion of all endometrial cancer patients would have such mutations; however, in a number of studies addressing this question, extremely few germline mutations have been found. Summarising the studies by Katabuchi et al ,6Kobayashi et al ,7 Lim et al ,8 Gurin et al ,9 and Kowalski et al ,10 only one germline mutation (in MLH1 ) was found in a total of 352 EC patients (0.3%). In these studies, mutations were sought in all patients whose tumours were microsatellite instability (MSI) positive. Recent reports have suggested that MSH6 might account for many endometrial cancers and that families with these mutations show atypical features of HNPCC with endometrial and ovarian cancers outnumbering colorectal cancers.11 12Additionally, MSI, a hallmark of HNPCC, was low in most tumours associated with MSH6 mutations or was preferentially shown by mononucleotide repeats rather than dinucleotide repeats.12-14 Previous studies have reported that 9-25% of sporadic endometrial cancers display microsatellite instability .7 9 15-18 In the majority of cases, this instability arises through hypermethylation of the MLH1 promoter region.9 19-21 This epigenetic change results …

The problem of detecting and/or isolating faults in dynamical systems is assessed. In contrast to previous approaches, the residual vector is considered to be a design variable as a free transfer function in addition to the actual filter which is supposed to minimize the residual. Some main directions are suggested, and a numerical algorithm implementing part of these is proposed.

In this paper we describe a new approach to contour extraction and tracking, which is based on the principles of active contour models, yet it overcomes its shortcomings. We formally introduce active rays, describe the contour extraction as an e nergy minimization problem and discuss what active contours and active rays have in common.The main difference is that for active rays a unique ordering of the contour elements in the 2D image plane is given, which cannot be found for active conto urs. This is advantageous for pred icting the contour elements' position and prevents crossings in the contour. Furthermore, another advantage of this approach is that instead of an energy minimization in the 2D image plane the minimization is reduced to a 1D search problem. The approach a lso shows any-time behavior which is important with respect to real- time applications. Finally, the method allows for the management of multiple hypotheses of the object's boundary.First results on real image sequences show the suitability of this approach for real- time object tracking. The contour tracking can be done within the image frame rate (25 fps) on standard Unix workstations (HP 735).