Oligosaccharyltransferase structures provide novel insight into the mechanism of asparagine-linked glycosylation in prokaryotic and eukaryotic cells

Abstract

Asparagine-linked (N-linked) glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring upon the majority of proteins that enter the secretory pathway. X-ray crystal structures of the single subunit OSTs from eubacterial and archaebacterial organisms revealed the location of donor and acceptor substrate binding sites and provided the basis for a catalytic mechanism. Cryoelectron microscopy structures of the octameric yeast OST provided substantial insight into the organization and assembly of the multisubunit oligosaccharyltransferases. Furthermore, the cryoelectron microscopy structure of a complex consisting of a mammalian OST complex, the protein translocation channel and a translating ribosome revealed new insight into the mechanism of cotranslational glycosylation.

Keywords: N-glycosylation; OST structure; cotranslational glycosylation; lipid-linked oligosaccharide; oligosaccharyltransferase.

Fig. 1.

Subunit composition, membrane topology and domain organization of the STT3A and STT3B complexes. Multisubunit OSTs are composed of three subcomplexes. Proteins are shown using their mammalian names, with yeast protein names shown in parentheses. Yeast lack homologs of DC2 and KCP2. The C-terminal four-helix bundle of RPN1 and the N-terminal domain of RPN2 are not present in the yeast homologs (Ost1 and Swp1, respectively). NTD, MD and TRX designate N-terminal domains, middle domains and the thioredoxin domain, and are based upon the cryo-EM structure of the yeast OST (Bai et al. 2018; Wild et al. 2018) and the structures of OST6 and TUSC3 (Schulz et al. 2009; Mohorko et al. 2014). The diagrams for STT3A and STT3B depict the location of EL1, EL5 and a well-ordered N-glycan that is important for STT3 function.

Fig. 2.

Structure of the eukaryotic oligosaccharyltransferases. (A) High-resolution structure of the yeast OST viewed from the plane of the membrane with the ER lumenal domain at the top. Subunits are color coded as in Figure 1 and are labeled. The blue star is positioned near the WWD motif. N-glycans are shown as black sticks. (B) Interaction between the STT3A complex and the RNC-Sec61 complex shown in the same orientation as in panel A. OST subunits are color coded as in Figure 1. The lumenal loop of DC2 interacts with the C-terminal tails of Sec61β (marine) and Sec61γ (light magenta). Lumenal domains of RPN1, RPN2, OST48 and TMEM258 are not shown. The C-terminal four-helix bundle of RPN1 interacts with 60 S ribosomal subunit protein eL28 and 28 S rRNA helices H25 and H19/20. The WWD motif in STT3A is shown as magenta spheres. An unidentified TM span near Sec61 is designated by an asterisk. (C, D) Structural homology between TMs 2-4 of OST3 (C) and DC2 (D) as viewed from the plane of the membrane. N and C-termini are labeled, where N of OST3 corresponds to residue S216. The figure was made with PYMOL v2.1software and PDB files 6FTI (B) and 6EZN (A, C).

Fig. 3.

Oligosaccharyltransferase in metazoan and fungal organisms. (A) Formation of complexes between the oligosaccharyltransferase and the RNC-Sec61 complex is controlled by the presence of positive determinants in the STT3A complex and negative determinants in the STT3B complex. For simplicity, the diagrams do not show RPN2, OST48, DAD1, TMEM258, KCP2 and OST4. (B) STT3A cotranslationaly glycosylates proteins using a scanning mechanism. A skipped sequon is indicated by a red asterisk. (C) STT3B can mediate cotranslational or posttranslocational glycosylation of acceptor sites that were skipped by STT3A before the protein folds. (D) By analogy to the mammalian STT3B complex we propose that the yeast OST glycosylates substrates by a mechanism that does not rely on a direct interaction with the Sec61-RNC complex. For simplicity, we do not depict glycoproteins that are translocated by the heptameric Sec complex or the Ssh1p complex.