LacdiNAc synthase B4GALNT3 has a unique PA14 domain and suppresses N-glycan capping.

Structural variation of N-glycans is essential for the regulation of glycoprotein functions. GalNAcß1-4GlcNAc (LacdiNAc or LDN), a unique subterminal glycan structure synthesized by B4GALNT3 or B4GALNT4, is involved in the clearance of N-glycoproteins from the blood and maintenance of cell stemness. Such regulation of glycoprotein functions by LDN is largely different from that by the dominant subterminal structure, N-acetyllactosamine (Galß1-4GlcNAc, LacNAc). However, the mechanisms by which B4GALNT activity is regulated and how LDN plays different roles from LacNAc remain unclear. Here, we found that B4GALNT3 and four have unique domain organization containing a noncatalytic PA14 domain, which is a putative glycan-binding module. A mutant lacking this domain dramatically decreases the activity toward various substrates, such as N-glycan, O-GalNAc glycan, and glycoproteins, indicating that this domain is essential for enzyme activity and forms part of the catalytic region. In addition, to clarify the mechanism underlying the functional differences between LDN and LacNAc, we examined the effects of LDN on the maturation of N-glycans, focusing on the related glycosyltransferases upstream and downstream of B4GALNT. We revealed that, unlike LacNAc synthesis, prior formation of bisecting GlcNAc in N-glycan almost completely inhibits LDN synthesis by B4GALNT3. Moreover, the presence of LDN negatively impacted the actions of many glycosyltransferases for terminal modifications, including sialylation, fucosylation, and human natural killer-1 synthesis. These findings demonstrate that LDN has significant impacts on N-glycan maturation in a completely different way from LacNAc, which could contribute to obtaining a comprehensive overview of the system regulating complex N-glycan biosynthesis.

Crystal structure of the complex of CLEC12A and an antibody that interferes with binding of diverse ligands.

C-type lectin receptors (CLRs) are a family of pattern recognition receptors, which detect a broad spectrum of ligands via small carbohydrate-recognition domains (CRDs). CLEC12A is an inhibitory CLR that recognizes crystalline structures such as monosodium urate crystals. CLEC12A also recognizes mycolic acid, a major component of mycobacterial cell walls, and suppresses host immune responses. Although CLEC12A could be a therapeutic target for mycobacterial infection, structural information on CLEC12A was not available. We report here the crystal structures of human CLEC12A (hCLEC12A) in ligand-free form and in complex with 50C1, its inhibitory antibody. 50C1 recognizes human-specific residues on the top face of hCLEC12A CRD. A comprehensive alanine scan demonstrated that the ligand-binding sites of mycolic acid and monosodium urate crystals may overlap with each other, suggesting that CLEC12A utilizes a common interface to recognize different types of ligands. Our results provide atomic insights into the blocking and ligand-recognition mechanisms of CLEC12A and leads to the design of CLR-specific inhibitors.

The stem region of α1,6-fucosyltransferase FUT8 is required for multimer formation but not catalytic activity.

Alpha-1,6-fucosyltransferase (FUT8) synthesizes core fucose in N-glycans, which plays critical roles in various physiological processes. FUT8, as with many other glycosyltransferases, is a type-II membrane protein, and its large C-terminal catalytic domain is linked to the FUT8 stem region, which comprises two α-helices. Although the stem regions of several glycosyltransferases are involved in the regulation of Golgi localization, the functions of the FUT8 stem region have not been clarified as yet. Here, we found that the FUT8 stem region is essential for enzyme oligomerization. We expressed FUT8Δstem mutants, in which the stem region was replaced with glycine/serine linkers, in FUT8-KO HEK293 cells. Our immunoprecipitation and native-PAGE analysis showed that FUT8 WT formed a multimer but FUT8Δstem impaired multimer formation in the cells, although the mutants retained specific activity. In addition, the mutant protein had lower steady-state levels, increased endoplasmic reticulum localization, and a shorter half-life than FUT8 WT, suggesting that loss of the stem region destabilized the FUT8 protein. Furthermore, immunoprecipitation analysis of another mutant lacking a part of the stem region revealed that the first helix in the FUT8 stem region is critical for multimer formation. Our findings demonstrated that the FUT8 stem region is essential for multimer formation but not for catalytic activity, providing insights into how the FUT8 protein matures and functions in mammalian cells.

Examination of differential glycoprotein preferences of N-acetylglucosaminyltransferase-IV isozymes a and b.

The N-glycans attached to proteins contain various GlcNAc branches, the aberrant formation of which correlates with various diseases. N-Acetylglucosaminyltransferase-IVa (GnT-IVa or MGAT4A) and Gnt-IVb (or MGAT4B) are isoenzymes that catalyze the formation of the ß1,4-GlcNAc branch in N-glycans. However, the functional differences between these isozymes remain unresolved. Here, using cellular and UDP-Glo enzyme assays, we discovered that GnT-IVa and GnT-IVb have distinct glycoprotein preferences both in cells and in vitro. Notably, we show that GnT-IVb acted efficiently on glycoproteins bearing an N-glycan premodified by GnT-IV. To further understand the mechanism of this reaction, we focused on the noncatalytic C-terminal lectin domain, which selectively recognizes the product glycans. Replacement of a nonconserved amino acid in the GnT-IVb lectin domain with the corresponding residue in GnT-IVa altered the glycoprotein preference of GnT-IVb to resemble that of GnT-IVa. Our findings demonstrate that the C-terminal lectin domain regulates differential substrate selectivity of GnT-IVa and GnT-IVb, highlighting a new mechanism by which N-glycan branches are formed on glycoproteins.

N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates.

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) catalyzes the formation of an N-glycan ß1,6-GlcNAc branch on selective target proteins in the Golgi apparatus and is involved in cancer malignancy and autoimmune disease etiology. Several three-dimensional structures of GnT-V were recently solved, and the recognition mechanism of the oligosaccharide substrate was clarified. However, it is still unclear how GnT-V selectively acts on glycoprotein substrates. In this study, we focused on an uncharacterized domain at the N-terminal side of the luminal region (N domain) of GnT-V, which was previously identified in a crystal structure, and aimed to reveal its role in GnT-V action. Using lectin blotting and fluorescence assisted cell sorting analysis, we found that a GnT-VΔN mutant lacking the N domain showed impaired biosynthetic activity in cells, indicating that the N domain is required for efficient glycosylation. To clarify this mechanism, we measured the in vitro activity of purified GnT-VΔN toward various kinds of substrates (oligosaccharide, glycohexapeptide, and glycoprotein) using HPLC and a UDP-Glo assay. Surprisingly, GnT-VΔN showed substantially reduced activity toward the glycoprotein substrates, whereas it almost fully maintained its activity toward the oligosaccharides and the glycopeptide substrates. Finally, docking models of GnT-V with substrate glycoproteins suggested that the N domain could interact with the substrate polypeptide directly. Our findings suggest that the N domain of GnT-V plays a critical role in the recognition of glycoprotein substrates, providing new insights into the mechanism of substrate-selective biosynthesis of N-glycans.

The SH3 domain in the fucosyltransferase FUT8 controls FUT8 activity and localization and is essential for core fucosylation.

Core fucose is an N-glycan structure synthesized by α1,6-fucosyltransferase 8 (FUT8) localized to the Golgi apparatus and critically regulates the functions of various glycoproteins. However, how FUT8 activity is regulated in cells remains largely unclear. At the luminal side and uncommon for Golgi proteins, FUT8 has an Src homology 3 (SH3) domain, which is usually found in cytosolic signal transduction molecules and generally mediates protein-protein interactions in the cytosol. However, the SH3 domain has not been identified in other glycosyltransferases, suggesting that FUT8's functions are selectively regulated by this domain. In this study, using truncated FUT8 constructs, immunofluorescence staining, FACS analysis, cell-surface biotinylation, proteomics, and LC-electrospray ionization MS analyses, we reveal that the SH3 domain is essential for FUT8 activity both in cells and in vitro and identified His-535 in the SH3 domain as the critical residue for enzymatic activity of FUT8. Furthermore, we found that although FUT8 is mainly localized to the Golgi, it also partially localizes to the cell surface in an SH3-dependent manner, indicating that the SH3 domain is also involved in FUT8 trafficking. Finally, we identified ribophorin I (RPN1), a subunit of the oligosaccharyltransferase complex, as an SH3-dependent binding protein of FUT8. RPN1 knockdown decreased both FUT8 activity and core fucose levels, indicating that RPN1 stimulates FUT8 activity. Our findings indicate that the SH3 domain critically controls FUT8 catalytic activity and localization and is required for binding by RPN1, which promotes FUT8 activity and core fucosylation.

Structural insight into the recognition of pathogen-derived phosphoglycolipids by C-type lectin receptor DCAR.

The C-type lectin receptors (CLRs) form a family of pattern recognition receptors that recognize numerous pathogens, such as bacteria and fungi, and trigger innate immune responses. The extracellular carbohydrate-recognition domain (CRD) of CLRs forms a globular structure that can coordinate a Ca2+ ion, allowing receptor interactions with sugar-containing ligands. Although well-conserved, the CRD fold can also display differences that directly affect the specificity of the receptors for their ligands. Here, we report crystal structures at 1.8-2.3 Å resolutions of the CRD of murine dendritic cell-immunoactivating receptor (DCAR, or Clec4b1), the CLR that binds phosphoglycolipids such as acylated phosphatidyl-myo-inositol mannosides (AcPIMs) of mycobacteria. Using mutagenesis analysis, we identified critical residues, Ala136 and Gln198, on the surface surrounding the ligand-binding site of DCAR, as well as an atypical Ca2+-binding motif (Glu-Pro-Ser/EPS168-170). By chemically synthesizing a water-soluble ligand analog, inositol-monophosphate dimannose (IPM2), we confirmed the direct interaction of DCAR with the polar moiety of AcPIMs by biolayer interferometry and co-crystallization approaches. We also observed a hydrophobic groove extending from the ligand-binding site that is in a suitable position to interact with the lipid portion of whole AcPIMs. These results suggest that the hydroxyl group-binding ability and hydrophobic groove of DCAR mediate its specific binding to pathogen-derived phosphoglycolipids such as mycobacterial AcPIMs.

Structural Aspects of Carbohydrate Recognition Mechanisms of C-Type Lectins.

Carbohydrate recognition is an essential function occurring in all living organisms. Lectins are carbohydrate-binding proteins and are classified into several families. In mammals, Ca2+-dependent C-type lectins, such as ß-galactoside-binding galectin and sialic acid-binding siglec, play crucial roles in the immune response and homeostasis. C-type lectins are abundant and diverse in animals. Their immunological activities include lymphocyte homing, pathogen recognition, and clearance of apoptotic bodies. C-type lectin domains are composed of 110-130 amino acid residues with highly conserved structural folds. Remarkably, individual lectins can accept a wide variety of sugar ligands and can distinguish subtle structural differences in closely related ligands. In addition, several C-type lectin-like proteins specifically bind to carbohydrate ligands in Ca2+-independent ways. The accumulated 3D structural evidence clarifies the unexpected structural versatility of C-type lectins underlying the variety of ligand binding modes. In this issue, we focus on the structural aspects of carbohydrate recognition mechanisms of C-type lectins and C-type lectin-like proteins.

3D Structure and Function of Glycosyltransferases Involved in N-glycan Maturation.

Glycosylation is the most ubiquitous post-translational modification in eukaryotes. N-glycan is attached to nascent glycoproteins and is processed and matured by various glycosidases and glycosyltransferases during protein transport. Genetic and biochemical studies have demonstrated that alternations of the N-glycan structure play crucial roles in various physiological and pathological events including progression of cancer, diabetes, and Alzheimer's disease. In particular, the formation of N-glycan branches regulates the functions of target glycoprotein, which are catalyzed by specific N-acetylglucosaminyltransferases (GnTs) such as GnT-III, GnT-IVs, GnT-V, and GnT-IX, and a fucosyltransferase, FUT8s. Although the 3D structures of all enzymes have not been solved to date, recent progress in structural analysis of these glycosyltransferases has provided insights into substrate recognition and catalytic reaction mechanisms. In this review, we discuss the biological significance and structure-function relationships of these enzymes.

Differential Labeling of Glycoproteins with Alkynyl Fucose Analogs.

Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions.

Analysis of protein landscapes around N-glycosylation sites from the PDB repository for understanding the structural basis of N-glycoprotein processing and maturation.

Asparagine-linked glycans (N-glycans) are attached onto nascent glycoproteins in the endoplasmic reticulum (ER) and subsequently processed by a set of processing enzymes in the ER and Golgi apparatus. Accumulating evidence has shown that not all N-glycans on glycoproteins are uniformly processed into mature forms (hybrid and complex types in mammals) through the ER and Golgi apparatus, and a certain set of glycans remains unprocessed as an "immature" form (high-mannose type in mammals). Much attention has been paid to environmental factors regulating N-glycoprotein maturation, such as the expression levels of glycosyltransferases/glycosidases. On the other hand, the influence of the 3D structure of the carrier glycoprotein on N-glycan maturation has been investigated mostly using individual model glycoproteins. To obtain more insights into N-glycoprotein maturation, we herein analyze glycoprotein structures extracted from the Protein Data Bank. We confirm that site-specific N-glycan processing is largely explained by the solvent accessibility of the glycosylated Asn residue and of the conjugated N-glycan. Potential bias of protein structural features toward immature or mature forms was explored within a range of concentric circles of fully folded glycoproteins. There does appear to be bias in amino acid composition and secondary structure. Most notably, γ-branched amino acid residues (Asn+Asp+Leu) occur more frequently on unstructured loop regions of immature glycoproteins. Structural features of the protein surface around the N-glycosylated site do seem to affect N-glycan processing and maturation.

3D structural analysis of protein O-mannosyl kinase, POMK, a causative gene product of dystroglycanopathy.

Orchestration of the multiple enzymes engaged in O-mannose glycan synthesis provides a matriglycan on α-dystroglycan (α-DG) which attracts extracellular matrix (ECM) proteins such as laminin. Aberrant O-mannosylation of α-DG leads to severe congenital muscular dystrophies due to detachment of ECM proteins from the basal membrane. Phosphorylation at C6-position of O-mannose catalyzed by protein O-mannosyl kinase (POMK) is a crucial step in the biosynthetic pathway of O-mannose glycan. Several mis-sense mutations of the POMK catalytic domain are known to cause a severe congenital muscular dystrophy, Walker-Warburg syndrome. Due to the low sequence similarity with other typical kinases, structure-activity relationships of this enzyme remain unclear. Here, we report the crystal structures of the POMK catalytic domain in the absence and presence of an ATP analogue and O-mannosylated glycopeptide. The POMK catalytic domain shows a typical protein kinase fold consisting of N- and C-lobes. Mannose residue binds to POMK mainly via the hydroxyl group at C2-position, differentiating from other monosaccharide residues. Intriguingly, the two amino acid residues K92 and D228, interacting with the triphosphate group of ATP, are donated from atypical positions in the primary structure. Mutations in this protein causing muscular dystrophies can now be rationalized.

Biophysical Analyses for Probing Glycan-Protein Interactions.

Glycan-protein interactions occur at many physiological events, and the analyses are of considerable importance for understanding glycan-dependent mechanisms. Biophysical approaches including 3D structural analysis are essential for revealing glycan-protein interactions at the atomic level. The inherent diversity of glycans suits them to function as identification tags, e.g., distinguish self from the nonself components of pathogens. However, the complexity of glycans and poor affinities for interaction partners limit the usefulness of conventional analyses. To cope with such troublesome glycans, a logical sequence of biophysical analyses need to be developed. In this chapter, we introduce a workflow of glycan-protein interaction analysis consisting of six steps: preparation of lectin and glycan, screening of glycan ligand, determination of binding epitope, quantitative interaction analysis, 3D structural analysis, and molecular dynamics simulation. Our increasing knowledge and understanding of lectin-glycan interactions will hopefully lead to the design of glyco-based medicines and vaccines.

Crystallographic analysis of murine p24γ2 Golgi dynamics domain.

The p24 family proteins form homo- and hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. It consists of four subfamilies (p24α, p24ß, p24γ, and p24δ). p24γ2 plays crucial roles in the selective transport of glycosylphosphatidylinositol-anchored proteins. Here, we determined the crystal structure of mouse p24γ2 Golgi dynamics (GOLD) domain at 2.8 Å resolution by the single anomalous diffraction method using intrinsic sulfur atoms. In spite of low sequence identity among p24 family proteins, p24γ2 GOLD domain assumes a ß-sandwich fold, similar to that of p24ß1 or p24δ1. An additional short α-helix is observed at the C-terminus of the p24γ2 GOLD domain. Intriguingly, p24γ2 GOLD domains crystallize as dimers, and dimer formation seems assisted by the short α-helix. Dimerization modes of GOLD domains are compared among p24 family proteins. Proteins 2017; 85:764-770. © 2016 Wiley Periodicals, Inc.

Distinct roles for each N-glycan branch interacting with mannose-binding type Jacalin-related lectins Orysata and Calsepa.

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-glycan-binding 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.

Enhancement of solubility and yield of a ß-glucan receptor Dectin-1 C-type lectin-like domain in Escherichia coli with a solubility-enhancement tag.

Dectin-1 is a C-type lectin-like pattern recognition receptor for ß(1-3)-glucans. It plays a crucial role in protecting against fungal invasion through binding to ß-glucans which are commonly present on the fungal cell wall. To probe its ligand binding mechanism by NMR, we expressed the recombinant murine Dectin-1 C-type lectin-like domain (CTLD) in E. coli using pCold vector and purified it. However, the high concentration of Dectin-1 CTLD required for NMR analysis could not be attained due to its inherent low solubility and low bacterial expression. In this study, we tried to increase expression and solubility of Dectin-1 CTLD by codon optimization and fusion of a GB1 tag (B1 domain of streptococcal Protein G). GB1 was inserted on either the N-terminal (NT) or C-terminal end as well as both terminal ends of human and mouse Dectin-1 CTLDs. A pure monomeric sample was only obtained with NT-GB1 fused mouse Dectin-1. Expression of mouse Dectin-1 CTLD yielded 0.9 ± 0.2 mg/L culture, codon optimized mouse Dectin-1 CTLD produced 1.4 ± 0.2 mg/L, and the tag-fused domain 7.1 ± 0.3 mg/L. The tag also increased solubility from 0.1 mM to 1.4 mM. The recombinant protein was correctly folded, in a monomeric state, and specifically bound ß-glucan laminarin. These results indicate that fusing GB1 to the N-terminus of mouse Dectin-1 domain advantageously increases yield and solubility, allows retention of native structure, and that the site of fusion is critical.

The α-helical region in p24γ2 subunit of p24 protein cargo receptor is pivotal for the recognition and transport of glycosylphosphatidylinositol-anchored proteins.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are group of proteins that depend on p24 cargo receptors for their transport from the endoplasmic reticulum to the Golgi apparatus. The GPI anchor is expected to act as a sorting and transport signal, but so far little is known about the recognition mechanism. In the present study we investigate the GPI-AP transport in cell knockdown of p24γ, the most diverse p24 subfamily. Knockdown of p24γ2 but not of other p24γ family members impaired the transport of a reporter GPI-AP. Restoration of the knockdown-induced phenotype using chimeric constructs between p24γ2 and the related p24γ1 further implied a role of the α-helical region of p24γ2 but not its GOLD domain in the specific binding of GPI-APs. We conclude that motifs in the membrane-adjacent α-helical region of p24γ2 are involved in recognition of GPI-APs and are consequently responsible for the incorporation of these proteins into coat protein complex II-coated transport vesicles.

Crystal structure of anti-polysialic acid antibody single chain Fv fragment complexed with octasialic acid: insight into the binding preference for polysialic acid.

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.

Recognition of bisecting N-acetylglucosamine: structural basis for asymmetric interaction with the mouse lectin dendritic cell inhibitory receptor 2.

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.

Crystal structures of carbohydrate recognition domain of blood dendritic cell antigen-2 (BDCA2) reveal a common domain-swapped dimer.

We report on crystal structures of a carbohydrate recognition domain (CRD) of human C-type lectin receptor blood dendritic cell antigen-2 (BDCA2). Three different crystal forms were obtained at 1.8-2.3 Å resolution. In all three, the CRD has a basic C-type lectin fold, but a long loop extends away from the core domain to form a domain-swapped dimer. The structures of the dimers from the three different crystal forms superimpose well, indicating that domain swapping and dimer formation are energetically stable. The structure of the dimer is compared with other domain-swapped proteins, and a possible regulation mechanism of BDCA2 is discussed.