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

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.

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

“…In the previously reported crystal CtUGGT structures, a full length eukaryotic UGGT revealed four thioredoxin-like domains (TRXL1-4) arranged in a long arc, terminating in two β-sandwiches (βS1 and βS2) tightly clasping the glucosyltransferase family 24 (GT24) domain [5]. The wild-type protein was captured in three different conformations, called 'open' (PDB ID 5MZO), 'intermediate' (PDB ID 5MU1) and 'closed' (PDB ID 5N2J).…”

“…Only correctly folded glycoproteins escape UGGT-mediated reglucosylation and can progress down the secretory pathway -to the Golgi and beyond. More than 25 years after the discovery of UGGT [3], recent structural and functional work has uncovered the protein's multi-domain architecture and obtained preliminary evidence of its inter-domain conformational flexibility [4][5][6]. Negativestain electron microscopy and small-angle X-ray scattering (SAXS) first revealed an arc-like structure with some degree of structural variability [4].…”

“…Negativestain electron microscopy and small-angle X-ray scattering (SAXS) first revealed an arc-like structure with some degree of structural variability [4]. Soon after, four distinct full length Chaetomium thermophilum UGGT (CtUGGT) crystal structures, together with a 15 Å cryo-EM reconstruction of the same protein, suggested that the C-terminal portion -comprising two β-sandwiches and the catalytic domainconstitutes a relatively rigid structure, while most of the conformational flexibility localizes to the four N-terminal thioredoxin-like (TRXL) domains [5]. Contrary to these observations, relative flexibility of the catalytic domain with respect to the rest of the structure was proposed by a different study of Thermomyces dupontii UGGT (TdUGGT), based on atomic force microscopy data and a 25 Å negative stain EM map, to which crystal structures for the catalytic domain and the remaining part of TdUGGT were separately fitted [6,7].…”

Target highlights from the first post‐PSI CASP experiment (CASP12, May–August 2016)

“…To aid our understanding of UGGT structure and function, we determined four distinct crystal structures of Chaetomium thermophilum UGGT, aka Ct UGGT . An unexpected structural feature of the UGGT molecule is the unusual subdomain structure of the first thioredoxin‐like domain (TRXL1), encoded by residues 43–216 in Ct UGGT.…”

“…104,105 To aid our understanding of UGGT structure and function, we determined four distinct crystal structures of Chaetomium thermophilum UGGT, aka CtUGGT. 106 An unexpected structural feature of the UGGT molecule is the unusual subdomain structure of the first thioredoxinlike domain (TRXL1), encoded by residues 43-216 in CtUGGT. The published sequence-based secondary structure predictions in this region was rather accurate, with most helices and sheets correctly predicted from sequence-but the UGGT TRXL1 domain boundaries were not well predicted.…”