The flagellum of Methanococcus voltae is composed of four structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDS-polyacrylamide gel indicating significant posttranslational modification. We describe here the structural characterization of the flagellin glycan from M. voltae using mass spectrometry to examine the proteolytic digests of the flagellin proteins in combination with NMR analysis of the purified glycan using a sensitive, cryogenically cooled probe. Nano-liquid chromatography-tandem mass spectrometry analysis of the proteolytic digests of the flagellin proteins revealed that they are post-translationally modified with a novel Nlinked trisaccharide of mass 779 Da that is composed of three sugar residues with masses of 318, 258, and 203 Da, respectively. In every instance the glycan is attached to the peptide through the asparagine residue of a typical N-linked sequon. The glycan modification has been observed on 14 of the 15 sequon sites present on the four flagellin structural proteins. The novel glycan structure elucidated by NMR analysis was shown to be a trisaccharide composed of -ManpNAcA6Thr-(1-4)--GlcpNAc3NAcA-(1-3)--GlcpNAc linked to Asn. In addition, the same trisaccharide was identified on a tryptic peptide of the S-layer protein from this organism implicating a common N-linked glycosylation pathway.Glycosylation of prokaryotic proteins is now well accepted, and examples of N-and O-glycosylation and of attachment of glycosylphosphatidylinositol anchors can now be found in the literature (1, 2). However, in contrast to eukaryotic glycosylation systems, prokaryotic systems display considerable diversity in the structure of the respective glycans and the proximal monosaccharide linkage. As a consequence there is considerable interest in determining the structural and genetic basis of glycan production among these diverse prokaryotic systems.The archaeal flagellum is a unique motility structure that is distinct from the well characterized bacterial flagellum (3). In contrast to bacterial flagellar assembly where newly synthesized flagellin is incorporated at the distal tip of the filament, it is believed that mature archaeal flagellin is incorporated at the base of the filament. In recent studies, the assembly of archaeal flagellum has been shown to more closely resemble a second bacterial motility system, the type IV pilus, where the structural protein pilin is synthesized with an unusual signal peptide and a hydrophobic N terminus. In Archaea, signal peptidases have been shown to cleave a signal peptide of the preflagellin proteins to produce mature flagellin, which is then incorporated into the filament (4). Flagellated archaeal species have one to five flagellin genes organized into a fla locus (3). The marine archaeon Methanococcus voltae has four flagellin structural genes organized in two transcriptional units: one unit contains flaA, whereas the second unit contains flaB1, flaB...
Campylobacter jejuni is well known for synthesizing ganglioside mimics within the glycan component of its lipooligosaccharide (LOS), which have been implicated in triggering GuillainBarré syndrome. We now confirm that this pathogen is capable of synthesizing a much broader spectrum of host glycolipid/ glycoprotein mimics within its LOS. P blood group and paragloboside (lacto-N-neotetraose) antigen mimicry is exhibited by RM1221, a strain isolated from a poultry source. RM1503, a gastroenteritis-associated strain, expresses lacto-N-biose and sialyl-Lewis c units, the latter known as the pancreatic tumorassociated antigen, DU-PAN-2 (or LSTa). C. jejuni GC149, a Guillain-Barré syndrome-associated strain, expresses an unusual sialic acid-containing hybrid oligosaccharide with similarity to both ganglio and P k antigens and can, through phase variation of its LOS biosynthesis genes, display GT1a or GD3 ganglioside mimics. We show that the sialyltransferase CstII and the galactosyltransferase CgtD are involved in the synthesis of multiple mimic types, with LOS structural diversity achieved through evolving allelic substrate specificity.
UHRF1 is a key mediator of inheritance of epigenetic DNA methylation patterns during cell division and is a putative target for cancer therapy. Recent studies indicate that interdomain interactions critically influence UHRF1's chromatin-binding properties, including allosteric regulation of its histone binding. Here, using an integrative approach that combines small angle X-ray scattering, NMR spectroscopy, and molecular dynamics simulations, we characterized the dynamics of the tandem tudor domain–plant homeodomain (TTD–PHD) histone reader module, including its 20-residue interdomain linker. We found that the apo TTD–PHD module in solution comprises a dynamic ensemble of conformers, approximately half of which are compact conformations, with the linker lying in the TTD peptide–binding groove. These compact conformations are amenable to cooperative, high-affinity histone binding. In the remaining conformations, the linker position was in flux, and the reader adopted both extended and compact states. Using a small-molecule fragment screening approach, we identified a compound, 4-benzylpiperidine-1-carboximidamide, that binds to the TTD groove, competes with linker binding, and promotes open TTD–PHD conformations that are less efficient at H3K9me3 binding. Our work reveals a mechanism by which the dynamic TTD–PHD module can be allosterically targeted with small molecules to modulate its histone reader function for therapeutic or experimental purposes.
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