Sequence and Structural Determinants of Mannose Recognition

Mannose, an abundant cell surface monosaccharide binds to a diverse set of receptors, which are involved in a variety of important cellular processes. Structural analysis has been carried out on all the proteins containing non-covalently bound mannose as a monosaccharide in the Protein Data Bank, to identify common recognition principles. Proteins, highly specific to mannose, belonging to the super family of bulb lectins, are found to contain a consensus sequence motif QXDXNXVXY, which has been identified to be essential for mannose binding. Analysis of this motif in the crystal structures of bulb lectins has led to the understanding of the contribution of individual residues in mannose recognition. Comparison with other mannose binding proteins, reveals common hydrogen bonding patterns in all of them, despite differences in sequence, overall fold and the substructures at the binding sites of individual proteins. A database analysis also suggests that although the topology of the backbone, as at the binding site in bulb lectins, can generate mannose binding capability in a few other proteins, sequence and disposition of not only the residues in the motif, but also the residues in the neighborhood play a crucial role in allowing that property to be retained

Computational analysis of multivalency in lectins: structures of garlic lectin-oligosaccharide complexes and their aggregates

Multivalency in lectins is a phenomenon that has been discussed at considerable length. The structural basis for the role of multivalency in garlic lectin has been investigated here through computational studies. Biochemical studies have shown that the binding affinity of garlic lectin for high mannose oligosaccharides is orders of magnitude greater than that for mannose. Modeling and energy calculations clearly indicate that such increase in affinity cannot be accounted for by binding of these oligosaccharides at any of the six sites of a garlic lectin dimer. These studies also indicate that a given oligosaccharide cannot bind simultaneously to more than one binding site on a lectin dimer. The possibility of a given oligosaccharide simultaneously binding to and hence linking two or more lectin molecules was therefore explored. This study showed that trimannosides and higher oligomers can cross-link lectin dimers, amplifying the protein-oligosaccharide interactions severalfold, thus explaining the role of multivalency in enhancing affinity. A comprehensive exploration of all possible cross-links posed a formidable computational problem. Even a partial exploration involving a carefully chosen region of the conformational space clearly showed that a given dimer pair can be cross-linked not only by a single oligosaccharide molecule but also simultaneously by two oligosaccharides. The number of such possible double cross-links, including those forming interesting tetrameric structures, generally increases with the size of the oligosaccharide, correlating with the biochemical data. In addition to their immediate relevance to garlic lectin, these studies are of general interest in relation to lectin-oligosaccharide interactions

Re-refinement using reprocessed data to improve the quality of the structure: a case study involving garlic lectin

The structure of dimeric garlic lectin was previously determined to an effective resolution of 2.8 A using X-ray intensity data processed by the XDS package and refined using X-PLOR [Chandra et al. (1999), J. Mol. Biol. 285, 1157±1168]. Repeated attempts to grow better crystals with a view to improving the deÆnition of the structure did not succeed.The available raw data were then reprocessed using DENZO. The structure was re-refined with both X-PLOR and CNS separately using the reprocessed data, which extended to a resolution of 2.2 A . These two sets of refinements and the two sets using the XDS-processed data afforded an opportunity to compare the performance of different data-processing and refinement packages when dealing with data from weakly diffracting crystals. The best results were obtained when CNS was employed for refinement using data processed by DENZO. The quality and the resolution of the map and the definition of the structure improved substantially. In particular, the amino-acid residues at the variable locations in the sequence, and hence the isolectins, could be identified with a high degree of confidence. It could be established that the crystal asymmetric unit contains two identical heterodimers. The new refined structure also provided a better definition of other finer structural details

Computational analysis of multivalency in lectins: structures of garlic lectin± oligosaccharide complexes and their aggregates

Multivalency in lectins is a phenomenon that has been discussed at considerable length. The structural basis for the role of multivalency in garlic lectin has been investigated here through computational studies. Biochemical studies have shown that the binding affinity of garlic lectin for high mannose oligosaccharides is orders of magnitude greater than that for mannose. Modeling and energy calculations clearly indicate that such increase in affinity cannot be accounted for by binding of these oligosaccharides at any of the six sites of a garlic lectin dimer. These studies also indicate that a given oligosaccharide cannot bind simultaneously to more than one binding site on a lectin dimer. The possibility of a given oligosaccharide simultaneously binding to and hence linking two or more lectin molecules was therefore explored. This study showed that trimannosides and higher oligomers can cross-link lectin dimers, amplifying the protein±oligosaccharide interactions severalfold, thus explaining the role of multivalency in enhancing affinity. A comprehensive exploration of all possible cross-links posed a formidable computational problem. Even a partial exploration involving a carefully chosen region of the conformational space clearly showed that a given dimer pair can be cross-linked not only by a single oligosaccharide molecule but also simultaneously by two oligosaccharides. The number of such possible double crosslinks, including those forming interesting tetrameric structures, generally increases with the size of the oligosaccharide,correlating with the biochemical data. In addition to their immediate relevance to garlic lectin, these studies are of general interest in relation to lectin±oligosaccharide interactions

Crystal structure of the kinase domain of human protein tyrosine kinase 6 (PTK6) at 2.33 Å resolution

Human Protein tyrosine kinase 6 (PTK6) (EC:2.7.10.2), also known as the breast tumor kinase (BRK), is an intracellular non-receptor Src-related tyrosine kinase expressed in a majority of human breast tumors and breast cancer cell lines, but its expression is low or completely absent in normal mammary glands. In the recent past, several studies have suggested that PTK6 is a potential therapeutic target in cancer. To understand its structural and functional properties, the PTK6 kinase domain (PTK6-KD) gene was cloned, overexpressed in a baculo-insect cell system, purified and crystallized at room temperature. X-ray diffraction data to 2.33 Å resolution was collected on a single PTK6-KD crystal, which belonged to the triclinic space group P1. The Matthews coefficient calculation suggested the presence of four protein molecules per asymmetric unit, with a solvent content of ∼50.The structure has been solved by molecular replacement and crystal structure data submitted to the protein data bank under the accession number 5D7V. This is the first report of apo PTK6-KD structure crystallized in DFG-in and αC-helix-out conformation

Crystal structure of the kinase domain of human protein tyrosine kinase 6 (PTK6) at 2.33 Å resolution

Human Protein tyrosine kinase 6 (PTK6) (EC:2.7.10.2), also known as the breast tumor kinase (BRK), is an intracellular non-receptor Src-related tyrosine kinase expressed in a majority of human breast tumors and breast cancer cell lines, but its expression is low or completely absent in normal mammary glands. In the recent past, several studies have suggested that PTK6 is a potential therapeutic target in cancer. To understand its structural and functional properties, the PTK6 kinase domain (PTK6-KD) gene was cloned, overexpressed in a baculo-insect cell system, purified and crystallized at room temperature. X-ray diffraction data to 2.33 Å resolution was collected on a single PTK6-KD crystal, which belonged to the triclinic space group P1. The Matthews coefficient calculation suggested the presence of four protein molecules per asymmetric unit, with a solvent content of ∼50.The structure has been solved by molecular replacement and crystal structure data submitted to the protein data bank under the accession number 5D7V. This is the first report of apo PTK6-KD structure crystallized in DFG-in and αC-helix-out conformation

Crystal Structure of a Dimeric Mannose-specific Agglutinin from Garlic: Quaternary Association and Carbohydrate Specificity

A mannose-specific agglutinin, isolated from garlic bulbs, has been crystallized in the presence of a large excess of a-D-mannose, in space group C2 and cell dimensions, a=203.24, b=43.78, c=79.27 A, $\beta$ = 112.4 , with two dimers in the asymmetric unit. X-ray diffraction data were collected up to a nominal resolution of 2.4 A and the structure was solved by molecular replacement. The structure, refined to an R-factor of 22.6% and an R_f_r_e_e of 27.8% reveals a $\beta$-prism II fold, similar to that in the snowdrop lectin, comprising three antiparallel four-stranded $\beta$-sheets arranged as a 12-stranded $\beta$-barrel, with an approximate internal 3-fold symmetry. This agglutinin is, however, a dimer unlike snowdrop lectin which exists as a tetramer, despite a high degree of sequence similarity between them. A comparison of the two structures reveals a few substitutions in the garlic lectin which stabilise it into a dimer and prevent tetramer formation. Three mannose molecules have been identified on each subunit. In addition, electron density is observed for another possible mannose molecule per dimer resulting in a total of seven mannose molecules in each dimer. Although the mannose binding sites and the overall structure are similar in the subunits of snowdrop and garlic lectin, their specificities to glycoproteins such as GP120 vary considerably. These differences appear, in part, to be a direct consequence of the differences in oligomerisation, implying that variation in quaternary association may be a mode of achieving oligosaccharide specificity in bulb lectins

Armeringsutformningens effekt på sprickvidder i höga betongbalkar

Reinforced concrete deep beams are known for applications in tall buildings, foundations and offshore structures. Deep beams are structural elements with length and height within the same magnitude and have significantly smaller thickness compared to a conventional concrete beam. Deep beams in bending have non-linear strain distribution compared to conventional beams where Bernoulli’s hypothesis is valid. Crack formation is a common problem in reinforced concrete structures, which reduce the durability of the structure. Once the concrete cracks the tension reinforcement carry the tensile forces instead of the concrete. Therefore, the design of tension reinforcement is important since the serviceability should be retained even after the structure cracks. The crack widths can be limited by using proper reinforcement and one alternative is to combine tensile reinforcement with crack reinforcement.  The function of the reinforcement is to distribute the cracks over the cross section which leads to that many smaller cracks occur instead of fewer, wider cracks. Small cracks are seen as less of a problem compared to large cracks since larger cracks reduce the durability significantly. For deep beams, there is at the present no well-substantiated analysis model for how crack widths shall be calculated when having reinforcement in multiple layers with different diameters. The use of crack reinforcement in the outer bottom layer has by tradition been considered as a cost efficient way to achieve small crack widths. In this work the crack width in deep beams have been analysed using the finite element program Atena 2D. The numerical results have been verified by analytical calculations based on Eurocode 2. The aim is to achieve reduced crack widths  by analysing the combination of crack- and tensile reinforcement compared to the case with tensile reinforcement only. Tensile reinforcement has a larger diameter, for example ø25 mm, and crack reinforcement has smaller diameters, often between ø10 and ø16 mm. The result from the calculations with Atena showed that there was an improvement regarding the reduction of crack widths when using crack reinforcement in combination with tensile reinforcement compared to using tensile reinforcement only. However, this improvement decreased by using reinforcement in multiple layers since a tensile reinforcement bar 1ø25 mm needed to be replaced by approximately six crack reinforcement bars 6ø10 mm in order to achieve the same total reinforcement area. The main disadvantage was that more space was required to place all reinforcement bars in the cross section, which reduced the lever arm. The reduction of the lever arm resulted in a reduced capacity for the reinforcement and the cracks might unintentionally become wider than expected. Furthermore, significant reduction of both crack widths and reinforcement stresses were obtained when the total area for a case with 7ø25 mm was increased to 9ø25 mm. The increased total area of only tensile reinforcement ø25 mm reduced the crack width more compared to using a combination of crack- and tensile reinforcement, which could simplify the construction work at building sites and minimize time consumption.Armerade höga betongbalkar är kända för tillämpningar i höga byggnader, grundsulor och offshore konstruktioner. Höga balkar är konstruktionselement med längd och höjd i samma storleksordning och har betydligt mindre tjocklek jämfört med en konventionell betongbalk. Höga balkar i böjning har en icke-linjär töjningsfördelning jämfört med konventionella balkar där Bernoullis hypotes gäller. Sprickbildning är ett vanligt problem i armerade betongkonstruktioner, vilket minskar beständigheten hos konstruktionen. När betongbalken spricker kommer armeringen att ta upp dragkraften istället för betongen därför är utformningen av böjarmering viktig eftersom bruksgränstillståndet bör behållas även efter att konstruktionen spricker. Sprickvidderna kan begränsas genom att använda korrekt armering och ett alternativ är att kombinera kraftarmering med sprickarmering. Armeringens funktion är att sprida ut sprickorna över tvärsnittet vilket leder till att många små sprickor uppkommer i stället för färre, bredare sprickor. Små sprickor ses som ett mindre problem jämfört med stora sprickor eftersom större sprickor minskar beständigheten avsevärt. För höga balkar finns det för närvarande ingen välunderbyggd analysmodell för hur sprickvidder ska beräknas när de har armering i flera lager och med olika diametrar. Användningen av sprickarmering har traditionellt ansetts vara ett kostnadseffektivt sätt att uppnå små sprickvidder. I detta arbete har sprickvidden i höga balkar analyserats med hjälp av finita elementprogrammet Atena 2D. De numeriska resultaten har verifierats med analytiska beräkningar baserade på Eurokod 2. Syftet är att uppnå reducerade sprickvidder genom att analysera kombinationen av sprick- och kraftarmering jämfört med fallet med endast kraftarmering. Kraftarmeringen har en större diameter, till exempel ø25 mm och sprickarmering har mindre diametrar, ofta mellan ø10 och ø16 mm. Resultaten från beräkningarna i Atena visade att sprickvidderna minskade vid användning av sprickarmering i kombination med kraftarmering jämfört med användning av endast kraftarmering. Denna förbättring minskade emellertid i och med användning av armering i flera lager. En kraftarmeringsstång 1ø25 mm behöver ersättas med ungefär sex sprickarmeringsstänger, 6ø10 mm, för att uppnå samma totala armeringsarea. Den största nackdelen var att det krävdes mer utrymme för att placera alla sprickarmeringsstänger i tvärsnittet, vilket minskade hävarmen. Minskningen av hävarmen medförde en reducerad kapacitet i armeringen och sprickorna blev bredare än förväntat. Vidare erhölls signifikant reduktion av både sprickvidder och armeringsspänningar när den totala arean för ett fall med 7ø25 mm ökades till 9ø25 mm. Den ökade totalarean av endast kraftarmeringsstänger ø25 mm minskade sprickvidden mer jämfört med att använda en kombination av sprick- och kraftarmering vilket skulle kunna förenkla byggarbetet på byggarbetsplatser och minimera tidsförbrukningen

Structure of catechol 1,2-dioxygenase from Pseudomonas arvilla.

International audienceCatechol 1,2-dioxygenase was first studied by Hayaishi and colleagues in 1950. In 1967, catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 (PaCTD) was chosen as a model system for the catecholic intradiol dioxygenases due to its activity, stability and expression level. Here we report the 2.65 A structure of the betabeta isozyme of PaCTD. The structure supports the hypothesis first made by Vetting and Ohlendorf [The 1.8A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker, Struct. Fold. Des. 8 (2000) 429-440.] that the catechol 1,2-dioxygenases are lipid binding proteins. The 5 amino-terminal helices involved in dimerization and forming the lipid binding site are shown to be plastic in their positions and orientations. The sequence differences between the alpha and beta polypeptides are located at the part of the monomers distant from dimerization surface and thus permit the formation of the 3 isozymes (alphaalpha, alphabeta, and betabeta) of PaCTD. The reported inactivation by sulfhydryl-modifying reagents is explained by the structure. The 10-residue Helix F (residues 203-212) is proposed to be central in communicating between the lipid binding site and the active site