Single-particle electron microscopy structure of UDP-glucose:glycoprotein glucosyltransferase suggests a selectivity mechanism for misfolded proteins

Abstract

The enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) mediates quality control of glycoproteins in the endoplasmic reticulum by attaching glucose to N-linked glycan of misfolded proteins. As a sensor, UGGT ensures that misfolded proteins are recognized by the lectin chaperones and do not leave the secretory pathway. The structure of UGGT and the mechanism of its selectivity for misfolded proteins have been unknown for 25 years. Here, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the structure of UGGT from Drosophila melanogaster at 18-Å resolution. Three-dimensional reconstructions revealed a cagelike structure with a large central cavity. Particle classification revealed flexibility that precluded determination of a highresolution structure. Introduction of biotinylation sites into a fungal UGGT expressed in Escherichia coli allowed identification of the catalytic and first thioredoxin-like domains. We also used hydrogen-deuterium exchange mass spectrometry to map the binding site of an accessory protein, Sep15, to the first thioredoxin-like domain. The UGGT structural features identified suggest that the central cavity contains the catalytic site and is lined with hydrophobic surfaces. This enhances the binding of misfolded substrates with exposed hydrophobic residues and excludes folded proteins with hydrophilic surfaces. In conclusion, we have determined the UGGT structure, which enabled us to develop a plausible functional model of the mechanism for UGGT's selectivity for misfolded glycoproteins.

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

“…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].…”

“…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]. 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].…”

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

“…The lack of any obvious sequence homology of this portion of UGGT with proteins of known fold led to the creation of a UGGT‐specific protein fold family (Pfam family PF06427) which gathers all known eukaryotic UGGT N‐terminal sequences. The most recent secondary structure and domain boundary predictions for UGGT detected three thioredoxin‐like (TRXL) domains in this region . The canonical TRXL fold (Pfam family PF13848) comprises a thioredoxin fold (a four‐stranded β sheet sandwiched between three α‐ helices, TRX=βαβ − αββα Pfam family PF00085, red in Figure ), modified by the insertion of a 4‐helix subdomain (TRXL=βαβ − αααα − αββα blue in Figure ) …”

“…86,87 Asparagine-, isoleucineand phenylalanine-ladders are found in the core of each monomer, stabilizing the basic b-helical architecture of the monomer. The asparagine ladder (residues 193, 214, 248, and 291) is located right at the T1 turn, while the isoleucine (residues 68, 98, 134, 167, 311, 357) and phenylalanine (residues 103,139,172,195) ladders are found in the PB1 and PB2 sheets, respectively. A ladder containing isoleucines and a leucine (Ile84, Ile147, Ile179 and Leu125) is present in the PB3 sheet.…”

“…The most recent secondary structure and domain boundary predictions for UGGT detected three thioredoxin-like (TRXL) domains in this region. 102,103 The canonical TRXL fold (Pfam family PF13848) comprises a thioredoxin fold (a four-stranded b sheet sandwiched between three ahelices, TRX5bab 2 abba Pfam family PF00085, red in Figure 10), modified by the insertion of a 4-helix subdomain (TRXL5bab 2 aaaa 2 abba blue in Figure 10). 104,105 To aid our understanding of UGGT structure and function, we determined four distinct crystal structures of Chaetomium thermophilum UGGT, aka CtUGGT.…”