Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation

Abstract

Oligosaccharyltransferase transfers an oligosaccharide chain to the asparagine residues in proteins. The archaeal and eubacterial oligosaccharyltransferases are single subunit membrane enzymes, referred to as "AglB" (archaeal glycosylation B) and "PglB" (protein glycosylation B), respectively. Only one crystal structure of a fulllength PglB has been solved. Here we report the crystal structures of the full-length AglB from a hyperthermophilic archaeon, Archaeoglobus fulgidus. The AglB and PglB proteins share the common overall topology of the 13 transmembrane helices, and a characteristic long plastic loop in the transmembrane region. This is the structural basis for the formation of the catalytic center, consisting of conserved acidic residues coordinating a divalent metal ion. In one crystal form, a sulfate ion was bound next to the metal ion. This structure appears to represent a dolichol-phosphate binding state, and suggests the release mechanism for the glycosylated product. The structure in the other crystal form corresponds to the resting state conformation with the well-ordered plastic loop in the transmembrane region. The overall structural similarity between the distantly related AglB and PglB proteins strongly indicates the conserved catalytic mechanism in the eukaryotic counterpart, the STT3 (stauroporine and temperature sensitivity 3) protein. The detailed structural comparison provided the dynamic view of the N-glycosylation reaction, involving the conversion between the structured and unstructured states of the plastic loop in the transmembrane region and the formation and collapse of the Ser/Thr-binding pocket in the C-terminal globular domain.

Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation

“…Oligosaccharyl Transfer Assay-The recombinant AglB enzymes of the long paralog of P. furiosus (PfAglB-L) and the long paralog and one of the two short paralogs of A. fulgidus (AfAglB-L and AfAglB-S1) were expressed in Escherichia coli and purified to homogeneity in the presence of 1% (w/v) n-dodecyl ␤-D-maltoside (7,28,29). As alternatives to the recombinant AglB enzymes of P. calidifontis and S. solfataricus, the LLO-depleted membrane fractions that contained the endogenous AglB proteins were prepared for the oligosaccharyl transfer assay.…”

The atomic structure of a eukaryotic oligosaccharyltransferase complex

“…2b, 5c), was previously shown to be lethal when mutated to alanine in Stt3 40 . The corresponding W215A mutation in AglB reduced the activity 11 , and the PglB equivalent Y196 directly interact with LLO 12 . The lower half of this groove is lined with numerous hydrophobic residues and is occupied by phospholipid PL8 in our structure (Fig.…”

ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterialN-oligosaccharyltransferases

“…Alanine replacement of the aspartic acid-39 residue in B. cenocepacia ArnT (aspartic acid-32 in S. enterica ArnT) resulted in a non-functional protein that lost the ability to modify lipid A with L-Ara4N. The functional importance of the aspartic acid in the DEX motif was established for several glycosyltransferases including the human PIG-M protein (Maeda et al 2001), Mycobacterium smegmatis PimE/EmbC proteins (Morita et al 2006;Korkegian et al 2014) and Pyrococcus furiosus AglB protein (Matsumoto et al 2013), underscoring the requirement for acidic residues in the active site GT-C, as previously suggested (Franco and Rigden 2003). However, the alanine replacement of the glutamic acid presents in the DEX motif resulted in a non-functional S. enterica ArnT but did not affect the B. cenocepacia protein.…”

“…These domains present a globular conformation mainly by α-helical domain surrounded by β-strands. The C-terminal region has been demonstrated as the peptide substrate binding domain for PglB, EmbC, Corynebacterium glutamicum RptA and A. fulgidus AglB-Long (Birch et al 2009;Lizak et al 2011;Matsumoto et al 2013;Korkegian et al 2014). Remarkably, our in vitro binding assay reveals for the first time that the C-terminal domain of ArnT is involved in lipid A substrate binding.…”