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The Dscam gene gives rise to thousands of diverse cell surface Receptors (1) thought to provide homophilic and heterophilic recognition specificity for neuronal wiring (2-4) and immune response (5). Mutually [...]

Two one Angstrom resolution crystal structures of liver alcohol dehydrogenase in complex with NADH and two inhibitors named as dimethyl sulfoxide and isobutyramide are reported. It is found that both structures present a dynamic state of inhibition.

A structure determination has been carried out with a kinetic study of the steroid-converting isozyme of horse liver alcohol dehydrogenase (SS-ADH). The structure was found by carrying out molecular replacement using ethanol (EE-ADH) as the search model. It was seen that In the ternary complex with NAD(super.+)/NADH, the carboxyl group of cholic acid ligates to the active site zinc ion. Likely this contributes to the strong biding in the ternary NAD(super.+) complex.

Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N‐linked glycosylation, a key post‐translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N‐linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X‐ray crystallography or other methods. In particular, obtaining diffraction‐quality crystals of such proteins and their complexes often requires modification of both the type of glycosylation patterns and their extent. Here, we demonstrate the benefits of producing target glycoproteins in the GlycoDelete human embryonic kidney 293 cell line that has been engineered to produce N‐glycans as short glycan stumps comprising N‐acetylglucosamine, galactose and sialic acid. Protein fragments of human Down syndrome cell‐adhesion molecule and colony‐stimulating factor 1 receptor were obtained from the GlycoDelete cell line for crystallization. The ensuing reduction in the extent and complexity of N‐glycosylation in both protein molecules compared with alternative glycoengineering approaches enabled their productive deployment in structural studies by X‐ray crystallography. Furthermore, a third successful implementation of the GlycoDelete technology focusing on murine IL‐12B is shown to lead to N‐glycosylation featuring an immature glycan in diffraction‐quality crystals. It is proposed that the GlycoDelete cell line could serve as a valuable go‐to option for the production of homogeneous glycoproteins and their complexes for structural studies by X‐ray crystallography and cryo‐electron microscopy. [ABSTRACT FROM AUTHOR]

Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors, and co-factors for signaling. Recently, genetic studies suggested that netrin is involved in neuronal haptotaxis, which requires a reversible adhesion process. Structural data indicate that netrin can also mediate trans -adhesion between apposing cells decorated with its receptors on the condition that the auxiliary guidance cue draxin is present. Here, we propose that netrin is involved in conditional adhesion, a reversible and localized process that can contribute to cell adhesion and migration. We suggest that netrin-mediated adhesion and signaling are linked, and that local environmental factors in the ventricular zone, the floor plate, or other tissues coordinate its function. Structural studies reveal molecular details of netrin-mediated clustering of DCC and uncoordinated 5 (UNC5), suggesting how they switch between growth cone attraction and repulsion in axon guidance. Three separate DCC-binding sites have been identified on netrin, and each site can be targeted by competing receptors or cofactors. Environmental cofactors may affect netrin-mediated signaling and adhesion. Structural studies suggest that the auxiliary guidance cue draxin locally expressed in the spinal cord alters netrin–DCC interactions to promote adhesion and axonal fasciculation. We propose that netrin mediates conditional adhesion between cells decorated with netrin receptors. Conditional adhesion is a reversible and highly localized process that can facilitate fasciculation, haptotaxis, and downstream signaling. [ABSTRACT FROM AUTHOR]

Extracellular glutamyl endopeptidase from Bacillus intermedius (BIEP) is a chymotrypsinlike serine protease, the crystal structure is found for C222(sub 1) and C2 of BIEP to 1.5 and 1.75 angstrom resolution. Examination reveals that the crystal structure of glutamyl endopeptidase from BIEP has a direct relation between Zymogen activation and substrate charge compensation.

Summary: Late blowing defect (LBD) is a major cause of spoilage in cheeses, caused by the growth of Clostridium spp. in the cheese matrix. We investigated the application of CTP1L, a bacteriophage endolysin active against Clostridium tyrobutyricum, and its enzymatically active and cell wall‐binding domains (EAD and CBD) attached to green fluorescent protein (GFP) to detect dairy‐related Clostridium species by fluorescence microscopy. GFP‐CTP1L and GFP‐CBD demonstrated specificity for Clostridium spp. by labelling 15 and 17 of 20 Clostridium strains, respectively, but neither bound to other members of the cheese microbiota. However, GFP‐EAD did not label any Clostridium strain tested. Unexpectedly, GFP‐CTP1L and GFP‐CBD were also able to bind to clostridial spores. In addition, GFP‐CBD allowed us to visualize the vegetative cells of C. tyrobutyricum directly in the matrix of a LBD cheese. Site‐directed mutants of GFP‐CTP1L and GFP‐CBD were made to examine the amino acids involved in binding and oligomer formation. Oligomerization was not essential for binding, but specific mutations in the CBD which affected oligomer formation also affected binding and lytic activity. We conclude that GFP‐CTP1L and GFP‐CBD could be good biomarkers for rapid detection of Clostridium spores in milk, so measures can be taken for the prevention of LBD in cheese, and also provide effective tools to study the development of Clostridium populations during cheese ripening. [ABSTRACT FROM AUTHOR]

Small‐angle X‐ray scattering (SAXS) analysis of biomolecules is increasingly common with a constantly high demand for comprehensive and efficient sample quality control prior to SAXS experiments. As monodisperse sample suspensions are desirable for SAXS experiments, latest dynamic light scattering (DLS) techniques are most suited to obtain non‐invasive and rapid information about the particle size distribution of molecules in solution. A multi‐receiver four‐channel DLS system was designed and adapted at the BioSAXS endstation of the EMBL beamline P12 at PETRA III (DESY, Hamburg, Germany). The system allows the collection of DLS data within round‐shaped sample capillaries used at beamline P12. Data obtained provide information about the hydrodynamic radius of biological particles in solution and dispersity of the solution. DLS data can be collected directly prior to and during an X‐ray exposure. To match the short X‐ray exposure times of around 1 s for 20 exposures at P12, the DLS data collection periods that have been used up to now of 20 s or commonly more were substantially reduced, using a novel multi‐channel approach collecting DLS data sets in the SAXS sample capillary at four different neighbouring sample volume positions in parallel. The setup allows online scoring of sample solutions applied for SAXS experiments, supports SAXS data evaluation and for example indicates local inhomogeneities in a sample solution in a time‐efficient manner. Biological macromolecules with different molecular weights were applied to test the system and obtain information about the performance. All measured hydrodynamic radii are in good agreement with DLS results obtained by employing a standard cuvette instrument. Moreover, applying the new multi‐channel DLS setup, a reliable radius determination of sample solutions in flow, at flow rates normally used for size‐exclusion chromatography–SAXS experiments, and at higher flow rates, was verified as well. This study also shows and confirms that the newly designed sample compartment with attached DLS instrumentation does not disturb SAXS measurements. [ABSTRACT FROM AUTHOR]