Structural constraints for the Crh protein from solid-state NMR experiments

We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples

Inverting family GH156 sialidases define an unusual catalytic motif for glycosidase action

Sialic acids are a family of related sugars that play essential roles in many biological events intimately linked to cellular recognition in both health and disease. Sialidases are therefore orchestrators of cellular biology and important therapeutic targets for viral infection. Here, we sought to define if uncharacterized sialidases would provide distinct paradigms in sialic acid biochemistry. We show that a recently discovered sialidase family, whose first member EnvSia156 was isolated from hot spring metagenomes, defines an unusual structural fold and active centre constellation, not previously described in sialidases. Consistent with an inverting mechanism, EnvSia156 reveals a His/Asp active center in which the His acts as a Bronsted acid and Asp as a Bronsted base in a single-displacement mechanism. A pre-dominantly hydrophobic aglycone site facilitates accommodation of a variety of 2-linked sialosides; a versatility that offers the potential for glycan hydrolysis across a range of biological and technological platforms

Coronin-1A Links Cytoskeleton Dynamics to TCRαβ-Induced Cell Signaling

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of αβT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-κB (IκB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts αβT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages

Estudio de la producción de ácido itacónico con Aspergillus terreus de la cepa MJL05 con diferentes variables

La producción de ácido itacónico (AI) con Aspergillus terreus MJL05 se realizó en fermentación sumergida en lote en un biorreactor agitado para estudiar el efecto de la variación de las concentraciones de nitrógeno, fósforo y carbono en el medio de producción. El glicerol, subproducto del biodiesel, fue reportado como un sustrato eficiente para obtener altas productividades de AI. Este fue utilizado como única fuente de carbono. Las relaciones entre nutrientes, C:N 18, N:P 10,8 y C:P 195 fueron seleccionadas como las mejores para aumentar la concentración de AI de 11,0 a 27,6 g/l con un a productividad volumétrica de 0,192 g IA/l.h, y una productividad específica de 0,013 g IA/g biomasa.h. Los rendimientos del bioproceso obtenidos fueron de Yx:s 0,27 g p.s. biomasa/g sustrato; Yp:x 1,63 g IA/g p.s. biomasa y Yp:s 0,44 g IA/g sustrato, lo que permite asumir la factibilidad de usar esta cepa para la producción de AI.Itaconic acid (IA) production by Aspergillus terreus MJL05 strain was investigated in submerged batch fermentation in a stirred bioreactor to determine the effect of varying the nitrogen, phosphorous and carbon sources concentrations in the production medium. Glycerol, a biodiesel by-product was reported as an efficient substrate to achieve high itaconic acid productivities. This was used as the sole carbon source. The resulting C:N 18, N:P 10.8 and C:P 195 ratios were selected as the best and allowed to improve IA concentration from 11.0 to 27.6 g/l with a volumetric productivity of 0.192 g IA/l.h and a specific productivity 0.013 g IA/g biomass.h. Bioprocess yields, Yx:s 0.27 g d.w. biomass/g substrate; Yp:x 1.63 g IA/g d.w. biomass and Yp:s 0.44 g IA/g substrate, allowed to assume the feasibility of using this strain for IA production

Failure of endotoxin to increase nonspecific resistance to infection of lipopolysaccharide low-responder mice.

In vitro and in vivo responses to lipopolysaccharide (LPS) and various other bacterial immunostimulants were compared in c3H/He low-responder mice. The principal findings were as follows. (i) Their splenic lymphocytes were stimulated by various gram-negative mitogens such as an Escherichia coli peptidoglycan, a detoxified derivative of LPS, and even endotoxins extracted by trichloroacetic acid that are known to contain protein; spleen cells of these mice were also transformed by two other B-cell mitogens extracted from acid-fast organisms. (ii) Their macrophages were refractory to LPS and weakly responsive to a mycobacterial prepartion. (iii) LPS failed to elicit nonspecific resistance in these mice against Klebsiella pneumoniae infection. (iv) Endotoxin extracted by trichloroacetic acid and a mycobacterial preparation that could increase nonspecific resistance to infection in other strains did not protect C3H/He mice against a challenge by K. pneumoniae, although both prepartions could evoke nonspecific responses of B cells in this low-responder subline

Amylose chain behavior in an interacting context I. Influence of a nonchair ring on the maltose conformations

In the presence of steric constraints, flexible forms, i.e., skew (S), boat (B) or half-boat (H), were evoked from experimental data and conformational analyses by molecular mechanics calculations for glucopyranose rings of amylose fragments. This important case occurring, for example, in amylose-amylase complexes, requires careful analysis of these flexible ring forms prior to any further conformational study. The influence of a nonchair (flexible) form on the maltose conformation is systematically evaluated, with an appropriate strategy using 'Semirelaxed'' maps and comparing them with those obtained from already known chair-chair (C-4(1)-C-4(1)) maps. Therefore, new low-energy maltose conformations are described and classified from flexible-chair and chair-flexible maps. These conformations are well dispersed inside significantly larger contours in the (phi, psi) projection. In a second stage, the consequence of these flexible ring forms is discussed in terms of amylose propagation apart from these new maltose conformations. Two propagation parameters are defined (tau, Omega), related to the local curvature of the chain and the relative orientation of the two mean ring planes. Low-energy conformations of C-4(1)-C-4(1) maltose have almost the same curvature between the two rings, whereas their relative orientations have well-identified Omega values. On the contrary, the presence of one flexible conformations considerably increases the variation range of both propagation parameters. Thus, new low-energy conformations allow local curvatures yielding from almost perpendicular to linear pairs of glucopyranose rings with relative orientation coveting about three-fourths of the total domain. This description is essential to understand amylose conformations in catalytic site and subsites of the amylases.

Multiple crystal forms of endoglucanase CelD: signal peptide residues modulate lattice formation.

International audienceThe crystal structure of Clostridium thermocellum endoglucanase CelD revealed an extended NH2-terminal segment (involving residues from the putative leader peptide) sticking out from the enzyme core to interact with a symmetry related molecule through an intermolecular salt bridge (Lys38-Asp201). Enzymatic digestion of CelD with various proteases emphasized the flexibility of the NH2-segment in solution. Proteolytic removal of Lys38 or the substitution of bridge-forming residues by site-directed mutagenesis promoted crystal packing arrangements that differ from that of wild type CelD. Crystals of wild-type CelD (a = 99.3 A c = 191.8 A) are trigonal, space group P3(1)21, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CelD (a = 100.4 A, c = 248.7 A), of CelDK38M (a = 100.1 A, c = 248.4 A) and of papain-treated CelDD201A (a = 99.9 A, c = 250.0 A) are trigonal, space group P3(1)21, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated CelD (a = 100.0 A, c = 254.3 A) and of CelDD201A (a = 99.8 A, c = 254.7 A) are hexagonal, space group P6(1)22, with one molecule in the asymmetric unit (form C). Only chymotrypsin-treated CelD (which preserves both Lys38 and Asp201) can grow in crystal form A upon macroseeding, indicating that formation of the intermolecular salt bridge is critical for stability of this crystal form. Flexible NH2- and COOH-terminal peptide extensions were found to influence crystal nucleation, but not crystal growth. The crystal structures of papain-treated CelD and chymotrypsin-treated CelD, determined at 3.5 A resolution by molecular replacement techniques, demonstrate that a small change in molecular orientation promoted by Lys38 account for the differences between crystal forms B and C.The crystal structure of Clostridium thermocellum endoglucanase CelD revealed an extended NH2-terminal segment (involving residues from the putative leader peptide) sticking out from the enzyme core to interact with a symmetry related molecule through an intermolecular salt bridge (Lys38-Asp201). Enzymatic digestion of CelD with various proteases emphasized the flexibility of the NH2-segment in solution. Proteolytic removal of Lys38 or the substitution of bridge-forming residues by site-directed mutagenesis promoted crystal packing arrangements that differ from that of wild type CelD. Crystals of wild-type CelD (a = 99.3 A c = 191.8 A) are trigonal, space group P3(1)21, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CelD (a = 100.4 A, c = 248.7 A), of CelDK38M (a = 100.1 A, c = 248.4 A) and of papain-treated CelDD201A (a = 99.9 A, c = 250.0 A) are trigonal, space group P3(1)21, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated CelD (a = 100.0 A, c = 254.3 A) and of CelDD201A (a = 99.8 A, c = 254.7 A) are hexagonal, space group P6(1)22, with one molecule in the asymmetric unit (form C). Only chymotrypsin-treated CelD (which preserves both Lys38 and Asp201) can grow in crystal form A upon macroseeding, indicating that formation of the intermolecular salt bridge is critical for stability of this crystal form. Flexible NH2- and COOH-terminal peptide extensions were found to influence crystal nucleation, but not crystal growth. The crystal structures of papain-treated CelD and chymotrypsin-treated CelD, determined at 3.5 A resolution by molecular replacement techniques, demonstrate that a small change in molecular orientation promoted by Lys38 account for the differences between crystal forms B and C

Water-protein hydrogen exchange in the micro-crystalline protein Crh as observed by solid state NMR spectroscopy

We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T-2(')-selective H-1-C-13-C-13 correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T-2(') selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on C-13 linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates