http://journals.iucr.org/d/issues/2012/02/00/isscontsbdy.html Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. Papers on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Papers on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability. en Copyright (c) 2012 International Union of Crystallography 2012-02-01 International Union of Crystallography International Union of Crystallography http://journals.iucr.org urn:issn:0907-4449 Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. Papers on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Papers on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability. text/html Acta Crystallographica Section D: Biological Crystallography, Volume 68, Part 2, 2012 text monthly 1 2002-01-01T00:00+00:00 2 68 2012-02-01 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography 95 urn:issn:0907-4449 med@iucr.org February 2012 2012-02-01 http://journals.iucr.org/logos/rss10d.gif http://journals.iucr.org/d/issues/2012/02/00/isscontsbdy.html Still image http://scripts.iucr.org/cgi-bin/paper?xb5042 The outer membrane protein complex (BAM complex) plays an important role in outer membrane protein (OMP) assembly in Escherichia coli. The BAM complex includes the integral β-barrel protein BamA as well as four lipoproteins: BamB, BamC, BamD and BamE. One of these lipoproteins, BamD, is essential for the survival of Escherichia coli. The structure of BamD at 2.6 Å resolution shows that this lipoprotein is composed of ten α-helices that form five tetratricopeptide-repeat (TPR) motifs. The arrangement of the BamD motifs is similar to that in the periplasmic part of BamA. One of the ten α-helices, α10, which has been shown to be important for the assembly of the BAM complex, is located in the very C-terminal region of BamD. A deep groove between TPR domains 4 and 5 is also observed. This groove, as well as the surface around α10, may provide binding sites for other components of the BAM complex. The C-terminal region of BamD serves as a platform for interactions with other components of the BAM complex. The N-terminal region shares structural similarity to other proteins whose functions are related to assistance in or regulation of secretion. Therefore, this region is likely to play an important role in the insertion of other outer membrane proteins. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Dong, C. Hou, H.-F. Yang, X. Shen, Y.-Q. Dong, Y.-H. 2012-01-06 doi:10.1107/S0907444911051031 International Union of Crystallography BamD is part of the Escherichia coli outer membrane protein complex (BAM complex) and is essential for the survival of E. coli. The structure of BamD at 2.6 Å resolution shows that this lipoprotein is composed of ten α-helices that form five tetratricopeptide-repeat (TPR) motifs. EN Gram-negative bacteria BAM complex OMP POTRA domains TPR motif The outer membrane protein complex (BAM complex) plays an important role in outer membrane protein (OMP) assembly in Escherichia coli. The BAM complex includes the integral β-barrel protein BamA as well as four lipoproteins: BamB, BamC, BamD and BamE. One of these lipoproteins, BamD, is essential for the survival of Escherichia coli. The structure of BamD at 2.6 Å resolution shows that this lipoprotein is composed of ten α-helices that form five tetratricopeptide-repeat (TPR) motifs. The arrangement of the BamD motifs is similar to that in the periplasmic part of BamA. One of the ten α-helices, α10, which has been shown to be important for the assembly of the BAM complex, is located in the very C-terminal region of BamD. A deep groove between TPR domains 4 and 5 is also observed. This groove, as well as the surface around α10, may provide binding sites for other components of the BAM complex. The C-terminal region of BamD serves as a platform for interactions with other components of the BAM complex. The N-terminal region shares structural similarity to other proteins whose functions are related to assistance in or regulation of secretion. Therefore, this region is likely to play an important role in the insertion of other outer membrane proteins. text/html Structure of Escherichia coli BamD and its functional implications in outer membrane protein assembly text 2 68 2012-01-06 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 95 101 http://scripts.iucr.org/cgi-bin/paper?dw5007 Post-translational modifications involving ubiquitin regulate a wide range of biological processes including protein degradation, responses to DNA damage and immune signalling. Ubiquitin polymerizes into chains which may contain eight different linkage types; the ubiquitin C-terminal glycine can link to one of the seven lysine residues or the N-terminal amino group of methionine in the distal ubiquitin molecule. The latter head-to-tail linkage type, referred to as a linear ubiquitin chain, is involved in NF-κB activation through specific interactions with NF-κB essential modulator (NEMO). Here, a crystal structure of linear diubiquitin at a resolution of 2.2 Å is reported. Although the two ubiquitin moieties do not interact with each other directly, the overall structure adopts a compact but not completely closed conformation with a few intermoiety contacts. This structure differs from the previously reported extended conformation, which resembles Lys63-linked diubiquitin, suggesting that the linear polyubiquitin chain is intrinsically flexible and can adopt multiple conformations. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Rohaim, A. Kawasaki, M. Kato, R. Dikic, I. Wakatsuki, S. 2012-01-13 doi:10.1107/S0907444911051195 International Union of Crystallography A new crystal structure of linear diubiquitin adopts a compact conformation that differs from its previously reported extended conformation. EN linear ubiquitin chain diubiquitin ubiquitin-binding domains NF-κB essential modulator Post-translational modifications involving ubiquitin regulate a wide range of biological processes including protein degradation, responses to DNA damage and immune signalling. Ubiquitin polymerizes into chains which may contain eight different linkage types; the ubiquitin C-terminal glycine can link to one of the seven lysine residues or the N-terminal amino group of methionine in the distal ubiquitin molecule. The latter head-to-tail linkage type, referred to as a linear ubiquitin chain, is involved in NF-κB activation through specific interactions with NF-κB essential modulator (NEMO). Here, a crystal structure of linear diubiquitin at a resolution of 2.2 Å is reported. Although the two ubiquitin moieties do not interact with each other directly, the overall structure adopts a compact but not completely closed conformation with a few intermoiety contacts. This structure differs from the previously reported extended conformation, which resembles Lys63-linked diubiquitin, suggesting that the linear polyubiquitin chain is intrinsically flexible and can adopt multiple conformations. text/html Structure of a compact conformation of linear diubiquitin text 2 68 2012-01-13 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 102 108 http://scripts.iucr.org/cgi-bin/paper?kw5037 The first crystal structure of a member of peptaibol antibiotic subfamily 4, trichovirin I-4A (14 residues), has been determined by direct methods and refined at atomic resolution. The monoclinic unit cell has two molecules in the asymmetric unit. Both molecules assume a 310 right-handed helical conformation and are significantly bent. The molecules pack loosely along the crystallographic twofold axis, forming two large tunnels between symmetry-related molecules in which no ordered solvent could be located. Carbonyl O atoms which are not involved in intramolecular hydrogen bonding participate in close van der Waals interactions with apolar groups. The necessary amphipathicity for biological activity of peptaibols is not realised in the crystal structure. Hence, a structural change of trichovirin to an α-helical conformation is proposed for membrane integration and efficient water/ion transportation across the lipid bilayer. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Gessmann, R. Axford, D. Owen, R.L. Brückner, H. Petratos, K. 2012-01-06 doi:10.1107/S090744491105133X International Union of Crystallography The first structure of a subfamily 4 peptaibol antibiotic, trichovirin I-4A, is described at atomic resolution. The structure suggests a conformational transition to obtain the necessary amphiphilicity for membrane insertion and water/ion transport across the hydrophobic barrier. EN peptaibols peptide antibiotics transmembrane ion channels transition from 310-helix to α-helix The first crystal structure of a member of peptaibol antibiotic subfamily 4, trichovirin I-4A (14 residues), has been determined by direct methods and refined at atomic resolution. The monoclinic unit cell has two molecules in the asymmetric unit. Both molecules assume a 310 right-handed helical conformation and are significantly bent. The molecules pack loosely along the crystallographic twofold axis, forming two large tunnels between symmetry-related molecules in which no ordered solvent could be located. Carbonyl O atoms which are not involved in intramolecular hydrogen bonding participate in close van der Waals interactions with apolar groups. The necessary amphipathicity for biological activity of peptaibols is not realised in the crystal structure. Hence, a structural change of trichovirin to an α-helical conformation is proposed for membrane integration and efficient water/ion transportation across the lipid bilayer. text/html Four complete turns of a curved 310-helix at atomic resolution: the crystal structure of the peptaibol trichovirin I-4A in a polar environment suggests a transition to α-helix for membrane function text 2 68 2012-01-06 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 109 116 http://scripts.iucr.org/cgi-bin/paper?mh5051 Evectin-2 is a recycling endosomal protein involved in retrograde transport. Its primary sequence contains an N-terminal pleckstrin homology (PH) domain and a C-terminal hydrophobic region. The PH domain of evectin-2 can specifically bind phosphatidylserine, which is enriched in recycling endosomes, and plays an essential role in retrograde transport from recycling endosomes to the trans-Golgi network. The structure of human evectin-2 PH domain in complex with O-phospho-l-serine has recently been reported and demonstrates how the head group of phosphatidylserine is recognized. However, it was not possible to elucidate from the structure why evectin-2 cannot bind phosphatidic acid or phosphatidylethanolamine, which share a common moiety with phosphatidylserine. Here, the crystal structure at 1.75 Å resolution of an apo form of human evectin-2 PH domain, in which the ligand-binding site is free from crystal packing and is thus appropriate for comparison with the structure of the complex, is reported. Comparison between the structures of the apo form and the O-phospho-l-serine complex revealed ligand-induced conformational change evoked by interaction between the carboxyl moiety of the head group of phosphatidylserine and the main-chain N atom of Thr14. This structural change effectively explains the strict ligand specificity of the PH domain of human evectin-2. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Okazaki, S. Kato, R. Uchida, Y. Taguchi, T. Arai, H. Wakatsuki, S. 2012-01-13 doi:10.1107/S0907444911051626 International Union of Crystallography The 1.75 Å resolution X-ray crystallographic structure of human evectin-2 pleckstrin homology domain revealed ligand-induced conformational change. This structural change effectively explains the strict phospholipid binding specificity. EN evectin-2 pleckstrin homology domain phospholipid binding Evectin-2 is a recycling endosomal protein involved in retrograde transport. Its primary sequence contains an N-terminal pleckstrin homology (PH) domain and a C-terminal hydrophobic region. The PH domain of evectin-2 can specifically bind phosphatidylserine, which is enriched in recycling endosomes, and plays an essential role in retrograde transport from recycling endosomes to the trans-Golgi network. The structure of human evectin-2 PH domain in complex with O-phospho-l-serine has recently been reported and demonstrates how the head group of phosphatidylserine is recognized. However, it was not possible to elucidate from the structure why evectin-2 cannot bind phosphatidic acid or phosphatidylethanolamine, which share a common moiety with phosphatidylserine. Here, the crystal structure at 1.75 Å resolution of an apo form of human evectin-2 PH domain, in which the ligand-binding site is free from crystal packing and is thus appropriate for comparison with the structure of the complex, is reported. Comparison between the structures of the apo form and the O-phospho-l-serine complex revealed ligand-induced conformational change evoked by interaction between the carboxyl moiety of the head group of phosphatidylserine and the main-chain N atom of Thr14. This structural change effectively explains the strict ligand specificity of the PH domain of human evectin-2. text/html Structural basis of the strict phospholipid binding specificity of the pleckstrin homology domain of human evectin-2 text 2 68 2012-01-13 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 117 123 http://scripts.iucr.org/cgi-bin/paper?dz5241 Global radiation damage to 19 thaumatin crystals has been measured using dose rates from 3 to 680 kGy s−1. At room temperature damage per unit dose appears to be roughly independent of dose rate, suggesting that the timescales for important damage processes are less than ∼1 s. However, at T = 260 K approximately half of the global damage manifested at dose rates of ∼10 kGy s−1 can be outrun by collecting data at 680 kGy s−1. Appreciable sample-to-sample variability in global radiation sensitivity at fixed dose rate is observed. This variability cannot be accounted for by errors in dose calculation, crystal slippage or the size of the data sets in the assay. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Warkentin, M. Badeau, R. Hopkins, J.B. Mulichak, A.M. Keefe, L.J. Thorne, R.E. 2012-01-17 doi:10.1107/S0907444911052085 International Union of Crystallography Approximately half of global radiation damage to thaumatin crystals can be outrun at 260 K if data are collected in less than 1 s. EN radiation damage dose rate room temperature protein crystallography Global radiation damage to 19 thaumatin crystals has been measured using dose rates from 3 to 680 kGy s−1. At room temperature damage per unit dose appears to be roughly independent of dose rate, suggesting that the timescales for important damage processes are less than ∼1 s. However, at T = 260 K approximately half of the global damage manifested at dose rates of ∼10 kGy s−1 can be outrun by collecting data at 680 kGy s−1. Appreciable sample-to-sample variability in global radiation sensitivity at fixed dose rate is observed. This variability cannot be accounted for by errors in dose calculation, crystal slippage or the size of the data sets in the assay. text/html Global radiation damage at 300 and 260 K with dose rates approaching 1 MGy s−1 text 2 68 2012-01-17 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 124 133 http://scripts.iucr.org/cgi-bin/paper?mh5053 A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Björkelid, C. Bergfors, T. Unge, T. Mowbray, S.L. Jones, T.A. 2012-01-06 doi:10.1107/S0907444911052231 International Union of Crystallography The structure of M. tuberculosis DXR (also known as IspC) in complex with the antibiotic FR-900098, NADPH and manganese was determined. This new crystal form diffracts to the highest resolution (1.65 Å) reported to date for this enzyme from any species. EN tuberculosis DXR IspC MEP pathway A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity. text/html Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098 text 2 68 2012-01-06 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 134 143 http://scripts.iucr.org/cgi-bin/paper?dw5005 Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed by the lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303 CE1 and Gln360 CG is reported. Structures of TvNiRb in complexes with phosphate (1.45 Å resolution) and sulfite (1.8 Å resolution), the structure of TvNiR in a complex with nitrite (1.83 Å resolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr–Cys and Tyr–Gln bonds may be associated with changes in the pKa value of the catalytic tyrosine. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Trofimov, A.A. Polyakov, K.M. Tikhonova, T.V. Tikhonov, A.V. Safonova, T.N. Boyko, K.M. Dorovatovskii, P.V. Popov, V.O. 2012-01-13 doi:10.1107/S0907444911052632 International Union of Crystallography An unusual covalent bond between the side chains of the catalytic Tyr residue and Gln decreases the activity of cytochrome c nitrite reductase. The Tyr residue is a proton donor during the catalysis. EN multihaem proteins covalent bonds proton transport catalytic mechanism Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed by the lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303 CE1 and Gln360 CG is reported. Structures of TvNiRb in complexes with phosphate (1.45 Å resolution) and sulfite (1.8 Å resolution), the structure of TvNiR in a complex with nitrite (1.83 Å resolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr–Cys and Tyr–Gln bonds may be associated with changes in the pKa value of the catalytic tyrosine. text/html Covalent modifications of the catalytic tyrosine in octahaem cytochrome c nitrite reductase and their effect on the enzyme activity text 2 68 2012-01-13 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 144 153 http://scripts.iucr.org/cgi-bin/paper?cb5004 Kif7, a member of the kinesin 4 superfamily, is implicated in a variety of diseases including Joubert, hydrolethalus and acrocallosal syndromes. It is also involved in primary cilium formation and the Hedgehog signalling pathway and may play a role in cancer. Its activity is crucial for embryonic development. Kif7 and Kif27, a closely related kinesin in the same subfamily, are orthologues of the Drosophila melanogaster kinesin-like protein Costal-2 (Cos2). In vertebrates, they work together to fulfil the role of the single Cos2 gene in Drosophila. Here, the high-resolution structure of the human Kif7 motor domain is reported and is compared with that of conventional kinesin, the founding member of the kinesin superfamily. These data are a first step towards structural characterization of a kinesin-4 family member and of this interesting molecular motor of medical significance. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Klejnot, M. Kozielski, F. 2012-01-13 doi:10.1107/S0907444911053042 International Union of Crystallography The human Kif7 motor domain structure provides insights into a kinesin of medical significance. EN Kif7 Kif27 human kinesins Hedgehog signalling Kif7, a member of the kinesin 4 superfamily, is implicated in a variety of diseases including Joubert, hydrolethalus and acrocallosal syndromes. It is also involved in primary cilium formation and the Hedgehog signalling pathway and may play a role in cancer. Its activity is crucial for embryonic development. Kif7 and Kif27, a closely related kinesin in the same subfamily, are orthologues of the Drosophila melanogaster kinesin-like protein Costal-2 (Cos2). In vertebrates, they work together to fulfil the role of the single Cos2 gene in Drosophila. Here, the high-resolution structure of the human Kif7 motor domain is reported and is compared with that of conventional kinesin, the founding member of the kinesin superfamily. These data are a first step towards structural characterization of a kinesin-4 family member and of this interesting molecular motor of medical significance. text/html Structural insights into human Kif7, a kinesin involved in Hedgehog signalling text 2 68 2012-01-13 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 154 159 http://scripts.iucr.org/cgi-bin/paper?mn5007 Rhizobial NodZ α1,6-fucosyltransferase (α1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5′-diphosphate-β-l-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signalling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two α1,6-FucT structures have been determined, both without any donor or acceptor molecule that could highlight the structural background of the catalytic mechanism. Here, the first crystal structures of α1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which is a byproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35 Å resolution. The fucose residue is exposed to solvent and is disordered. The enzyme–product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-l-glucosamine (penta-NAG). The structure has been determined at 1.98 Å resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-terminal domain, which are conserved among α1,2-, α1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand βC2 and helix αC3. In addition, there is a shift of the αC3 helix itself upon GDP-Fuc binding. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Brzezinski, K. Dauter, Z. Jaskolski, M. 2012-01-06 doi:10.1107/S0907444911053157 International Union of Crystallography Crystal structures of the bacterial α1,6-fucosyltransferase NodZ in complex with GDP and GDP-fucose are presented. EN glycosyltransferases fucosyltransferases family GT-23 glycosyltransferases chitooligosaccharide fucosylation Nod-factor biosynthesis nodulation Nod factors legume–rhizobium symbiosis nitrogen fixation Rhizobial NodZ α1,6-fucosyltransferase (α1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5′-diphosphate-β-l-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signalling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two α1,6-FucT structures have been determined, both without any donor or acceptor molecule that could highlight the structural background of the catalytic mechanism. Here, the first crystal structures of α1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which is a byproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35 Å resolution. The fucose residue is exposed to solvent and is disordered. The enzyme–product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-l-glucosamine (penta-NAG). The structure has been determined at 1.98 Å resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-terminal domain, which are conserved among α1,2-, α1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand βC2 and helix αC3. In addition, there is a shift of the αC3 helix itself upon GDP-Fuc binding. text/html Structures of NodZ α1,6-fucosyltransferase in complex with GDP and GDP-fucose text 2 68 2012-01-06 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 160 168 http://scripts.iucr.org/cgi-bin/paper?dz5239 The crystal structure of an RNA/DNA hybrid dodecamer, r(5′-uaaaagaaaagg):d(5′-CCTTTTCTTTTA), which contains three-quarters of the polypurine tract (PPT) sequence of the HIV RNA genome is reported. The hybrid structure was determined at 1.6 Å resolution and was found to have the A-form conformation. However, the presence of alternate conformations along the RNA template strand indicated increased flexibility of the PPT sequence. Two segments (at nucleotides 1–2 and 6–8) of the RNA chain have two conformations exhibiting differences in torsion and pseudorotation angles. For conformation I(1–2, 6–8), 25% of the RNA sugars have the C2′-exo pucker and the rest have the expected C3′-endo pucker. The II1–2 and II6–8 conformations of the RNA strand have one sugar with the C2′-exo pucker. None of the ribose rings exist in the C2′-endo form, in contrast to a previous report which postulated a C2′-endo ribose as a key structural element of the PPT. The widths of the minor groove for conformations I(1–2, 6–8) and II(1–2, 6–8) of the RNA strand are 9.2–10.5 and 9.4–10.7 Å, respectively. Both ranges are very close to the intervals accepted for A-form RNA duplexes. On the opposing DNA primer strand most of the sugars are C3′-endo, except for the 3′-terminal sugars, which are C2′-endo (T22) or O4′-endo (T23 and A24). The duplex includes a noncanonical u1(anti)·A24(syn) base interaction with only one hydrogen bond between the bases. This noncanonical base interaction at the 5′-end of the template distorts the values of the helical parameters of the adjacent base pair. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Drozdzal, P. Michalska, K. Kierzek, R. Lomozik, L. Jaskolski, M. 2012-01-06 doi:10.1107/S0907444911053327 International Union of Crystallography The high-resolution crystal structure of an RNA/DNA dodecamer with the HIV-1 polypurine-tract sequence reveals an A-type hybrid duplex with a complete absence of any C2′-endo ribonucleotides and with segments of increased flexibility along the RNA chain. EN HIV-1 polypurine tract RNA/DNA hybrid reverse transcription The crystal structure of an RNA/DNA hybrid dodecamer, r(5′-uaaaagaaaagg):d(5′-CCTTTTCTTTTA), which contains three-quarters of the polypurine tract (PPT) sequence of the HIV RNA genome is reported. The hybrid structure was determined at 1.6 Å resolution and was found to have the A-form conformation. However, the presence of alternate conformations along the RNA template strand indicated increased flexibility of the PPT sequence. Two segments (at nucleotides 1–2 and 6–8) of the RNA chain have two conformations exhibiting differences in torsion and pseudorotation angles. For conformation I(1–2, 6–8), 25% of the RNA sugars have the C2′-exo pucker and the rest have the expected C3′-endo pucker. The II1–2 and II6–8 conformations of the RNA strand have one sugar with the C2′-exo pucker. None of the ribose rings exist in the C2′-endo form, in contrast to a previous report which postulated a C2′-endo ribose as a key structural element of the PPT. The widths of the minor groove for conformations I(1–2, 6–8) and II(1–2, 6–8) of the RNA strand are 9.2–10.5 and 9.4–10.7 Å, respectively. Both ranges are very close to the intervals accepted for A-form RNA duplexes. On the opposing DNA primer strand most of the sugars are C3′-endo, except for the 3′-terminal sugars, which are C2′-endo (T22) or O4′-endo (T23 and A24). The duplex includes a noncanonical u1(anti)·A24(syn) base interaction with only one hydrogen bond between the bases. This noncanonical base interaction at the 5′-end of the template distorts the values of the helical parameters of the adjacent base pair. text/html Structure of an RNA/DNA dodecamer corresponding to the HIV-1 polypurine tract at 1.6 Å resolution text 2 68 2012-01-06 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 169 175 http://scripts.iucr.org/cgi-bin/paper?dz5240 In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similarity to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector l-arabinose has been determined at 2.2 Å resolution. The l-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the Kd value was 8.4 ± 0.4 µM. The effect of l-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Procházková, K. Čermáková, K. Pachl, P. Sieglová, I. Fábry, M. Otwinowski, Z. Řezáčová, P. 2012-01-17 doi:10.1107/S090744491105414X International Union of Crystallography The crystal structure of the effector-binding domain of the transcriptional repressor AraR from B. subtilis in complex with the effector molecule (l-arabinose) was determined at 2.2 Å resolution. A detailed analysis of the crystal identified a dimer organization that is distinctive from that of other members of the GalR/LacI family. EN repressors dimerization effector binding isothermal titration calorimetry differential scanning fluorimetry dimeric interface In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similarity to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector l-arabinose has been determined at 2.2 Å resolution. The l-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the Kd value was 8.4 ± 0.4 µM. The effect of l-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family. text/html Structure of the effector-binding domain of the arabinose repressor AraR from Bacillus subtilis text 2 68 2012-01-17 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 176 185 http://scripts.iucr.org/cgi-bin/paper?dz5245 Multi-copper oxidases constitute a family of proteins that are capable of coupling the one-electron oxidation of four substrate equivalents to the four-electron reduction of dioxygen to two molecules of water. The main catalytic stages occurring during the process have already been identified, but several questions remain, including the nature of the protonation events that take place during the reductive cleavage of dioxygen to water. The presence of a structurally conserved acidic residue (Glu498 in CotA laccase from Bacillus subtilis) at the dioxygen-entrance channel has been reported to play a decisive role in the protonation mechanisms, channelling protons during the reduction process and stabilizing the site as a whole. A second acidic residue that is sequentially conserved in multi-copper oxidases and sited within the exit channel (Asp116 in CotA) has also been identified as being important in the protonation process. In this study, CotA laccase has been used as a model system to assess the role of Asp116 in the reduction process of dioxygen to water. The crystal structures of three distinct mutants, D116E, D116N and D116A, produced by site-saturation mutagenesis have been determined. In addition, theoretical calculations have provided further support for a role of this residue in the protonation events. Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 Silva, C.S. Damas, J.M. Chen, Z. Brissos, V. Martins, L.O. Soares, C.M. Lindley, P.F. Bento, I. 2012-01-17 doi:10.1107/S0907444911054503 International Union of Crystallography The role of Asp116 in the pronotation events taking place during CotA laccase catalytic mechanism was investigated. The crystal structure determination of three distinct mutants (D116A, D116N and D116E), produced by site-saturation mutagenesis, together with theoretical calculations have provided evidence of its importance during the reductive cleavage of dioxygen to water. EN laccases trinuclear cluster dioxygen reduction protonation simulations electrostatic calculations Multi-copper oxidases constitute a family of proteins that are capable of coupling the one-electron oxidation of four substrate equivalents to the four-electron reduction of dioxygen to two molecules of water. The main catalytic stages occurring during the process have already been identified, but several questions remain, including the nature of the protonation events that take place during the reductive cleavage of dioxygen to water. The presence of a structurally conserved acidic residue (Glu498 in CotA laccase from Bacillus subtilis) at the dioxygen-entrance channel has been reported to play a decisive role in the protonation mechanisms, channelling protons during the reduction process and stabilizing the site as a whole. A second acidic residue that is sequentially conserved in multi-copper oxidases and sited within the exit channel (Asp116 in CotA) has also been identified as being important in the protonation process. In this study, CotA laccase has been used as a model system to assess the role of Asp116 in the reduction process of dioxygen to water. The crystal structures of three distinct mutants, D116E, D116N and D116A, produced by site-saturation mutagenesis have been determined. In addition, theoretical calculations have provided further support for a role of this residue in the protonation events. text/html The role of Asp116 in the reductive cleavage of dioxygen to water in CotA laccase: assistance during the proton-transfer mechanism text 2 68 2012-01-17 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography research papers 186 193 http://scripts.iucr.org/cgi-bin/paper?me0456 Copyright (c) 2012 International Union of Crystallography urn:issn:0907-4449 IUCr 2012-01-17 doi:10.1107/S0907444911042430 International Union of Crystallography EN Notes for authors text/html Notes for authors 2012 text 2 68 2012-01-17 Copyright (c) 2012 International Union of Crystallography Acta Crystallographica Section D: Biological Crystallography international union of crystallography 194 199