Inter-domain conformational flexibility underpins the activity of UGGT, the eukaryotic glycoprotein secretion checkpoint
Roversi P., Marti L., Caputo AT., Alonzi DS., Hill JC., Dent KC., Kumar A., Levasseur MD., Lia A., Waksman T., Basu S., Soto Albrecht Y., Qian K., McIvor JP., Lipp CB., Siliqi D., Vasiljević S., Mohammed S., Lukacik P., Walsh MA., Santino A., Zitzmann N.
Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170 kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively re-glucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER-retention, and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, the first crystal structures of a full length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc, which terminates in two β–sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β–sandwich and the fourth TRXL domain being encoded by non-consecutive stretches of sequence. In addition to the crystal structures, a 15 Å cryo-EM reconstruction reveals inter-domain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra inter-domain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and re-glucosylate its many different substrates, and/or enable re-glucosylation of N-linked glycans situated at variable distances from the site of misfold.