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The activity of glycosyltransferase GnT-V correlates with cancer malignancy and poor prognosis but its mechanism of action is poorly understood. Here, the authors solve crystal structures of free and substrate analog-bound GnT-V, providing insights into its catalytic mechanism and a basis for GnT-V inhibition.

Membrane proteins reside in the lipid bilayer of biomembranes and the structure and function of these proteins are closely related to their interactions with lipid molecules. Structural analyses of interactions between membrane proteins and lipids or detergents that constitute biological or artificial model membranes are important for understanding the functions and physicochemical properties of membrane proteins and biomembranes. Determination of membrane protein structures is much more difficult when compared with that of soluble proteins, but the development of various new technologies has accelerated the elucidation of the structure-function relationship of membrane proteins. This review summarizes the development of heavy atom derivative detergents and lipids that can be used for structural analysis of membrane proteins and their interactions with detergents/lipids, including their application with X-ray free-electron laser crystallography.

Self-propagating β-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes. [ABSTRACT FROM AUTHOR]

Mannose-binding type Jacalin-related lectins (mJRLs) bind to branched N-glycans via conserved sugar-binding sites. Despite, significant 3D structural similarities, each mJRL is known to have a unique binding preference toward various N-glycans. However, the molecular basis of varying binding preference is substantially unknown. Here, we report a detailed comparison of N-glycanbinding preference for two mJRLs, Orysata and Calsepa using frontal affinity chromatography (FAC), X-ray and molecular modeling. The FAC analysis using a panel of N-glycans shows difference in N-glycan-binding preference between the lectins. Orysata shows broader specificity toward most high-mannose-type glycans as well as biantennary complex-type glycans bearing an extension on the Manα1-6 branch. Whereas, Calsepa shows narrow specificity to complex-type glycans with bisecting GlcNAc. The X-ray crystallographic structure reveals that two Orysata lectins bind to one biantennary N-glycan (2:1 binding) where one lectin binds to mannose of the α1-3 branch, while the other interacts with an N-acetylglucosamine of the α1-6 branch. In contrast, Calsepa shows 1:1 binding where α1-3 branch and core chitobiose region N-glycan interacts with lectin, while α1-6 branch is flipped-back to the chitobiose core. Molecular dynamics study of Orysata bound to N-glycans substantiate possibility of two-binding modes for each N-glycan. Binding free energies calculated separately for α1-3 and α1-6 branches of each N-glycan suggest both branches can bind to Orysata. Overall these results suggest that each branch of N-glycan has a distinct role in binding to mJRLs and the nonbinding branch can contribute significantly to the binding affinity and hence to the specificity. [ABSTRACT FROM AUTHOR]

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein- coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams. [ABSTRACT FROM AUTHOR]

The 14-helix structure of oligo-β-peptides was significantly stabilized by direct attachment of CF3 groups to their backbones. Our studies indicate that this stabilization originates from the CF3-promoted increase in the intramolecular hydrogen-bonding ability of their back-bone amides, leading to a novel strategy to stabilize peptide folding. [ABSTRACT FROM AUTHOR]

The ubiquity of the sialic acid (2−3) galactose linkage in oligosaccharides of biological relevance necessitates a building block for the incorporation of this motif into oligosaccharides prepared by modular synthesis. The linear synthesis of the sialyl Lewis X tumor-associated antigen (1) has been accomplished in good yield using a sialic acid (2−3) galactose disaccharide building block. The disaccharide building block was synthesized efficiently from readily available galactal by a high-yielding and selective sialylation reaction. [ABSTRACT FROM AUTHOR]

ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related β-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to α-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Manα1-3Man and Glcβ1-3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using 15N-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through α-mannose-related recognition. [ABSTRACT FROM AUTHOR]

Dendritic cell inhibitory receptor 2 (DCIR2) is a C-type lectin expressed on classical dendritic cells. We recently identified the unique ligand specificity of mouse DCIR2 (mDCIR2) toward biantennary complex-type glycans containing bisecting N-acetylglucosamine (GlcNAc). Here, we report the crystal structures of the mDCIR2 carbohydrate recognition domain in unliganded form as well as in complex with an agalactosylated complex-type N-glycan unit carrying a bisecting GlcNAc residue. Bisecting GlcNAc and the α1-3 branch of the biantennary oligosaccharide asymmetrically interact with canonical and non-canonical mDCIR2 residues. Ligand-protein interactions occur directly through mDCIR2-characteristic amino acid residues as well as via a calcium ion and water molecule. Our structural and biochemical data elucidate for the first time the unique binding mode of mDCIR2 for bisecting GlcNAc-containing glycans, a mode that contrasts sharply with that of other immune C-type lectin receptors such as DC-SIGN. [ABSTRACT FROM AUTHOR]

Polysialic acid is a linear homopolymer of α2--8-linked sialic acids attached mainly onto glycoproteins. Cell surface polysialic acid plays roles in cell adhesion and differentiation events in a manner that is often dependent on the degree of polymerization (DP). Anti-oligo/polysialic acid antibodies have DP-dependent antigenic specificity, and such antibodies are widely utilized in biological studies for detecting and distinguishing between different oligo/polysialic acids. A murine monoclonal antibody mAb735 has a unique preference for longer polymers of polysialic acid (DP > 10), yet the mechanism of recognition at the atomic level remains unclear. Here, we report the crystal structure of mAb735 single chain variable fragment (scFv735) in complex with octasialic acid at 1.8 Å resolution. In the asymmetric unit, two scFv735 molecules associate with one octasialic acid. In both complexes of the unit, all the complementarity-determining regions except for L3 interact with three consecutive sialic acid residues out of the eight. A striking feature of the complex is that 11 ordered water molecules bridge the gap between antibody and ligand, whereas the direct antibody-ligand interaction is less extensive. The dihedral angles of the trisialic acid unit directly interacting with scFv735 are not uniform, indicating that mAb735 does not strictly favor the previously proposed helical conformation. Importantly, both reducing and nonreducing ends of the bound ligand are completely exposed to solvent.We suggest that mAb735 gains its apparent high affinity for a longer polysialic acid chain by recognizing every three sialic acid units in a paired manner. [ABSTRACT FROM AUTHOR]

Our previous studies on a β1,6-JV-acetylglucosaminyltrans-ferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward -linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodies-terase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular gly-cosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions. [ABSTRACT FROM AUTHOR]