Interdomain conformational flexibility underpins the activity of UGGT, the eukaryotic glycoprotein secretion checkpoint

DOI: 10.1073/pnas.1703682114

Authors: Pietro Roversi (Oxford Glycobiology Institute, University of Oxford) , Lucia Marti (Institute of Sciences of Food Production, Consiglio Nazionale delle Ricerche (CNR)) , Alessandro T. Caputo (Oxford Glycobiology Institute, University of Oxford) , Dominic S. Alonzi (Oxford Glycobiology Institute, University of Oxford) , Johan C. Hill (Oxford Glycobiology Institute, University of Oxford) , Kyle C. Dent (Diamond Light Source) , Abhinav Kumar (Oxford Glycobiology Institute, University of Oxford) , Mikail D. Levasseur (Oxford Glycobiology Institute, University of Oxford) , Andrea Lia (Oxford Glycobiology Institute, University of Oxford; Institute of Sciences of Food Production, Consiglio Nazionale delle Ricerche (CNR)) , Thomas Waksman (Oxford Glycobiology Institute, University of Oxford) , Souradeep Basu (Oxford Glycobiology Institute, University of Oxford) , Yentli Soto Albrecht (Oxford Glycobiology Institute, University of Oxford) , Kristin Qian (Oxford Glycobiology Institute, University of Oxford) , James Patrick Mcivor (Oxford Glycobiology Institute, University of Oxford) , Colette B. Lipp (Oxford Glycobiology Institute, University of Oxford) , Dritan Siliqi (Istituto di Cristallografia, Consiglio Nazionale delle Ricerche) , Snezana Vasilijevic (Oxford Glycobiology Institute, University of Oxford) , Shabaz Mohammed (Oxford Glycobiology Institute, University of Oxford) , Petra Lukacik (Diamond Light Source) , Martin A. Walsh (Diamond Light Source) , Angelo Santino (Institute of Sciences of Food Production, Consiglio Nazionale delle Ricerche (CNR)) , Nicole Zitzmann (Oxford Glycobiology Institute, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences Pnas

State: Published (Approved)
Published: June 2017

Abstract: 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 reglucosylates 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, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that 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 nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain 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 reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Diamond Offline Facilities: Electron Bio-Imaging Centre (eBIC)
Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , Krios I-Titan Krios I at Diamond

Other Facilities: BM14, ID30A-1 at ESRF

Added On: 25/07/2017 10:35

Documents:
1703682114.full.pdf

Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Microscopy Macromolecular Crystallography (MX) Electron Microscopy (EM) Cryo Electron Microscopy (Cryo EM)