Exploiting Topological Constraints To Reveal Buried Sequence Motifs in the Membrane-Bound N-Linked Oligosaccharyl Transferases

Abstract

The central enzyme in N-linked glycosylation is the oligosaccharyl transferase (OTase), which catalyzes glycan transfer from a polyprenyldiphosphate-linked carrier to select asparagines within acceptor proteins. PglB from Campylobacter jejuni is a single-subunit OTase with homology to the Stt3 subunit of the complex multimeric yeast OTase. Sequence identity between PglB and Stt3 is low (17.9%); however, both have a similar predicted architecture and contain the conserved WWDxG motif. To investigate the relationship between PglB and other Stt3 proteins, sequence analysis was performed using 28 homologs from evolutionarily distant organisms. Since detection of small conserved motifs within large membrane-associated proteins is complicated by divergent sequences surrounding the motifs, we developed a program to parse sequences according to predicted topology and then analyze topologically related regions. This approach identified three conserved motifs that served as the basis for subsequent mutagenesis and functional studies. This work reveals that several inter-transmembrane loop regions of PglB/Stt3 contain strictly conserved motifs that are essential for PglB function. The recent publication of a 3.4 Å-resolution structure of full-length C. lari OTase provides clear structural evidence that these loops play a fundamental role in catalysis The current study provides biochemical support for the role of the inter-transmembrane domain loops in OTase catalysis and demonstrates the utility of combining topology prediction and sequence analysis for exposing buried pockets of homology in large membrane proteins. The described approach allowed detection of the catalytic motifs prior to availability of structural data, and reveals additional catalytically relevant residues that are not predicted by structural data alone.

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

“…In accord with its critical role, the Glu residue is widely conserved in the TIXE (Thr-Ile-X-Glu) motif among archaea and eubacteria, and in the SVSE (Ser-Val-Ser-Glu) motif within eukaryotes (Fig. S4A), as mentioned previously (28). To assess the catalytic involvement of the metal-coordinating residues in AfAglB-L, we mutated these residues and measured the specific activities of the mutants (Fig.…”

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…salinarum was nonetheless modified with a sulfated glycan (173). While full-length sequence alignments failed to identify homology in the membrane-span- ning N-terminal regions of OSTs, Jaffee and Imperiali (199) recently developed a program that compared the sequences of the various OSTs in terms of a specific topographical feature, such as the first TMD or first loop region. With this approach, those authors were able to identify several conserved motifs in OSTs across all three domains.…”

“…The acidic residues in the two DxD motifs were shown in the crystal structure of C. lari PglB to be involved in the coordination of the divalent cation required for the activity of all OSTs (195). Using analysis of the sequence corresponding to topographical features of OSTs, a further motif was discovered in EL5, namely, [I/V]XXX[S/T][I/V]XE (199). The glutamic acid of this motif is thought, along with the conserved aspartic acid residues in ELs 1 and 3, to help form the pocket that accommodates the divalent cation and the nucleophilic asparagine.…”

“…The glutamic acid of this motif is thought, along with the conserved aspartic acid residues in ELs 1 and 3, to help form the pocket that accommodates the divalent cation and the nucleophilic asparagine. Mutation of the corresponding glutamic acid (E319) in PglB resulted in significantly lower or no activity, indicating the importance of this residue in OST activity (199). Analysis of the crystal structure of full-length AglB-L showed that D47 of the first DXD motif of EL1 (equivalent to C. lari PglB D56) and D161 and H163 of the second DXD motif of EL2 (C. lari D154 and D156) were all involved in the coordination of the divalent metal ion.…”

“…D47A and D47N mutations resulted in a complete loss of enzymatic activity, while a D47E mutant retaining the acidic charge still showed significant activity (20%). Interestingly, mutation of the corresponding residue in C. lari PglB (D54) to alanine, asparagine, or glutamic acid led to an inactive enzyme (199). E360A and E360Q mutations resulted in an inactive enzyme, although E360D showed 30% activity.…”