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X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk). Btk belongs to the Tec family of tyrosine kinases. Each member of the family contains five regions and mutations causing XLA have been isolated in all five regions. We have determined the solution structure of the Src homology 3 (SH3) domain of Btk using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on natural abundance and 15N-labeled protein material. The structure determination is complemented by investigation of backbone dynamics based on 15N NMR relaxation. The Btk SH3 forms a well-defined structure and shows the typical SH3 topology of two short antiparallel [Beta]-sheets packed almost perpendicular to each other in a sandwich-like fold. The N- and C-termini are more flexible as are peptide fragments in the RT and n-Src loops. The studied Btk SH3 fragment adopts two slowly interconverting conformations with a relative concentration ratio of 7:1. The overall fold of the minor form is similar to that of the major form, as judged on the basis of observed NOE connectivities and small chemical shift differences. A tryptophan (W251) ring flip is the favored mechanism for interconversion, although other possibilities cannot be excluded. The side chain of Y223, which becomes autophosphorylated upon activation of Btk, is exposed within the potential SH3 ligand binding site. Finally, we compare the present Btk SH3 structure with other SH3 structures.

The first synthesis of cyclic [beta]-1,3-heptaglucan from the reaction between 1,3(4)-[beta]-D-glucanase nucleophile mutant E115S of phanerochaete Chrysosporium Laminarinase 16A (Lam16A) and [alpha]-laminariheptaosyl fluoride ([alpha]L7F) is described. The study provides insight into the glcosynthase mechanism that joins the reducing and nonreducing ends of a single oligosaccharide.

A 1.8 Angstrom resolution three-dimensional structural determination of a complete Hypocrea jecorina glucoamylase is reported. The structural model with catalytic and starch binding domains traces the course of the 37-residue linker segment, serves to target the glucoamylase at sites where the starch granular matrix is disrupted and where the enzyme might most effectively function.

Glycosynthases are precise molecular instruments for making specifically linked oligosaccharides. X-ray crystallography screening of ligands bound to the 1,3(4)-β-o-glucanase nucleophile mutant E115S of Phanerochaete chtysosporium Laminarinase 16A (Lam16A) showed that laminariheptaose (L7) bound in an arch with the reducing and non reducing ends occupying either side of the catalytic cleft of the enzyme. The X-ray structure of Lam16A E115S in complex with α-laminariheptaosyl fluoride (αL7F) revealed how αL7F could make a nucleophilic attack upon itself. Indeed, when Lam16A E115S was allowed to react with αL7F the major product was a cyclic β-1,3-heptaglucan, as shown by mass spectrometry. NMR confirmed uniquely β-1,3-linkages and no reducing end. Molecular dynamics simulations indicate that the cyclic laminariheptaose molecule is not completely planar and that torsion angles at the glycosidic linkages fluctuate between two energy minima. This is the first report of a glycosynthase that joins the reducing and nonreducing ends of a single oligosaccharide and the first reported synthesis of cyclic β-glucan. [ABSTRACT FROM AUTHOR]

Amyloid nanofibrils are excellent scaffolds for designable materials that can be endowed with biotechnologically relevant functions. However, most of all excellent ideas and concepts that have been reported in the literature might never see real-world implementation in biotechnological applications. One bottleneck is the large-scale production of these materials. In this paper, we present an attempt to create a generic and scalable platform for producing ready-to-use functionalized nanofibrils directly from a eukaryotic organism. As a model material, we assembled Sup35(1-61) amyloid nanofibrils from Saccharomyces cerevisiae decorated with the Z-domain dimer, which has a high affinity toward antibody molecules. To this end, Komagataella pastoris was engineered by inserting gene copies of Sup35(1-61) and the protein chimera Sup35(1-61)-ZZ into the genome. This strain has the capability to constantly secrete amyloidogenic proteins into the extracellular medium, where the mature functionalized fibrils form, with a production yield of 35 mg/L culture. Another striking feature of this strategy is that the separation of the fibril material from the cells requires only centrifugation and resuspension in saline water. The fast production rates, minimal hands-on time, and high stability of the assembled material are some highlights that make the direct assembly of functionalized fibrils in the extracellular medium an alternative to production methods that are not suitable for large-scale production of designed amyloids.