Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a β-sheet of the membrane-distal ectodomain of SLAM using the side of its β-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their β-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

Mincle [macrophage inducible Ca2+ -dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6′-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor-TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle-citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca 2+ -dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.X-ray crystallography | innate immunity | mycobacteria | pattern-recognition receptors | myeloid cells

Asn-glycosylation is widespread not only in eukaryotes but also in archaea and some eubacteria. Oligosaccharyltransferase (OST) catalyzes the co-translational transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. Here, we report that a thermophilic archaeon, Pyrococcus furiosus OST is composed of the STT3 protein alone, and catalyzes the transfer of a heptasaccharide, containing one hexouronate and two pentose residues, onto peptides in an Asn-X-Thr/ Ser-motif-dependent manner. We also determined the 2.7-Å resolution crystal structure of the C-terminal soluble domain of Pyrococcus STT3. The structure-based multiple sequence alignment revealed a new motif, DxxK, which is adjacent to the well-conserved WWDYG motif in the tertiary structure. The mutagenesis of the DK motif residues in yeast STT3 revealed the essential role of the motif in the catalytic activity. The function of this motif may be related to the binding of the pyrophosphate group of lipidlinked oligosaccharide donors through a transiently bound cation. Our structure provides the first structural insights into the formation of the oligosaccharide-asparagine bond.

Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes Nglycosylation. We report the 2.8 Å resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria.Protein N-glycosylation is an important posttranslational modification that occurs in all domains of life (1). The enzyme that creates the oligosaccharide-asparagine linkage is oligosaccharyltransferase (OST).5 OST catalyzes the en bloc transfer of a preassembled oligosaccharide from a lipid carrier to asparagine residues in the glycosylation consensus (Asn-X-Thr/Ser, where X represents any amino acid except for Pro) of polypeptide chains (2-4). OST is a multisubunit membrane protein complex in higher eukaryotes. Yeast (Saccharomyces cerevisiae) OST consists of eight different subunits: Ost1p, Ost2p, Ost3p/ Ost6p, Ost4p, Wbp1, Swp1, Stt3p, and Ost5p (5), where Ost3p and Ost6p are paralogs that are present in two distinct OST isoforms (6). The cryoelectron microscopy structure of the digitonin-solubilized OST complex from yeast provided the relative arrangement of Ost1p, Wbp1, and Stt3p on the lumenal side of the complex (7,8).Stt3p is the catalytic subunit of the yeast OST enzyme (9). The vertebrate, insect, and plant equivalents are the two paralog proteins, STT3A and STT3B, which define distinct OST isoforms (10, 11). In lower eukaryotes, such as trypanosomatids, OST is a single-polypeptide membrane protein (3), and these single-subunit OST proteins consist of STT3 (staurosporine and temperature sensitivity 3) alone. In fact, the STT3s from Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major can each function as an OST enzyme when transferred into stt3-deficient yeast cells (12)(13)(14)(15). The prokaryotic OST is also a single-polypeptide protein. The STT3 homologs, PglB (protein glycosylation B) and AglB (archaeal glycosylation B), comprise the bacterial OST and the archaeal OST,. Multiple STT3/PglB/AglB paralogs also exist in some single-subunit OSTs. The trypanosomatids L. major and T. brucei contain four and three STT3 par...

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