Clamping, bending, and twisting inter-domain motions in the misfold-recognising portion of UDP-glucose:glycoprotein glucosyl-transferase

Abstract

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is the only known glycoprotein folding quality control checkpoint in the eukaryotic glycoprotein secretory pathway. When the enzyme detects a misfolded glycoprotein in the Endoplasmic Reticulum (ER), it dispatches it for ER retention by re-glucosylating it on one of its N-linked glycans. Recent crystal structures of a fungal UGGT have suggested the enzyme is conformationally mobile. Here, a negative stain electron microscopy reconstruction of UGGT in complex with a monoclonal antibody confirms that the misfold-sensing N-terminal portion of UGGT and its C-terminal catalytic domain are tightly associated. Molecular Dynamics (MD) simulations capture UGGT in so far unobserved conformational states, giving new insights into the molecule's flexibility. Principal component analysis of the MD trajectories affords a description of UGGT's overall inter-domain motions, highlighting three types of inter-domain movements: bending, twisting and clamping. These interdomain motions modify the accessible surface area of the enzyme's central saddle,likely enabling the protein to recognize and re-glucosylate substrates of different sizes and shapes, and/or re-glucosylate N-linked glycans situated at variable distances from the site of misfold. We propose to name "Parodi limit" the maximum distance between a site of misfolding on a UGGT glycoprotein substrate and an Nlinked glycan that monomeric UGGT can re-glucosylate on the same glycoprotein. MD simulations estimate the Parodi limit to be around 60-70 Å. Re-glucosylation assays using UGGT deletion mutants suggest that the TRXL2 domain is necessary for activity against urea-misfolded bovine thyroglobulin. Taken together, our findings support a "one-size-fits-all adjustable spanner" substrate recognition model, with a crucial role for the TRXL2 domain in the recruitment of misfolded substrates to the enzyme's active site.

Calnexin cycle – structural features of the ER chaperone system

“…The crystal structures show UGGT forms a saddle‐like shape with a large central cavity (Fig. 4B) [22,25]. The shape is consistent with the low‐resolution EM structures and with molecular envelope obtained in solution using SAXS data [22‐24].…”

“…Most likely, the cavity‐faced surfaces of UGGT TRXL2 and TRXL3 domains participate in recognition of misfolded substrates. The important role of TRXL2 in substrate reglucosylation has been recently supported by UGGT deletion mutagenesis and molecular dynamics simulations [25]. Future mutagenesis studies should confirm the substrate‐binding surfaces.…”

“…Early theories proposed a great deal of mobility between the N‐terminal part and catalytic domain, while the full‐length UGGT structures invariably showed the catalytic domain firmly entrenched in the β‐sandwich surface [22]. At the same time, negative‐stain EM and SAXS data implied significant movements of the catalytic domain in solution [24], but alternative interpretation is also possible [25]. Notably, the ability of catalytic domain to be stable in solution independently from the rest of UGGT is supported by crystal structures of individual catalytic domain [24].…”

“…This review provides an overview of proteins involved in calnexin cycle with an emphasis on recent structural insights (Table 1). These include calreticulin in the context of PLC, P‐domain recognition by ER chaperones, and structural characterization of UGGT [15,21‐25]. As recent structures of glucosidase II were well covered in other reviews [26,27], this work will focus on developments in understanding of UGGT and calreticulin and their mechanism of action.…”

Crocetin: A Systematic Review

“…GTs can specifically transfer sugar groups to receptor molecules (Modenutti et al, 2019). He et al applied to patent the preparation of crocetin glucose ester using glucose as the donor.…”