Abstract
In the endoplasmic reticulum (ER), a protein quality control system facilitates the efficient folding of newly synthesised proteins. In this system, a series of N-linked glycan intermediates displayed on the protein surface serve as quality tags. The ER folding-sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as a gatekeeper in the ER quality control system by specifically catalysing monoglucosylation onto incompletely folded glycoproteins, thereby enabling them to interact with lectin–chaperone complexes. Here we characterise the dynamic structure of this enzyme. Our crystallographic data demonstrate that the sensor region is composed of four thioredoxin-like domains followed by a β-rich domain, which are arranged into a C-shaped structure with a large central cavity, while the C-terminal catalytic domain undergoes a ligand-dependent conformational alteration. Furthermore, small-angle X-ray scattering, cryo-electron microscopy and high-speed atomic force microscopy have demonstrated that UGGT has a flexible modular structure in which the smaller catalytic domain is tethered to the larger folding-sensor region with variable spatial arrangements. These findings provide structural insights into the working mechanism whereby UGGT operates as a folding-sensor against a variety of glycoprotein substrates through its flexible modular structure possessing extended hydrophobic surfaces for the recognition of unfolded substrates.
Highlights
The endoplasmic reticulum (ER) possesses a sophisticated protein quality control system that ensures the appropriate folding and trafficking of newly synthesised proteins
Based on previous bioinformatic and crystallographic analyses, we proposed that the folding-sensor region harbours three tandem thioredoxin (Trx)-like domains followed by a β-strand-rich domain, displaying exposed hydrophobic patches putatively providing substrate-binding sites[12]
We characterised the overall structure and dynamic property of UDP-glucose:glycoprotein glucosyltransferase (UGGT) using an integrative approach by combining X-ray crystallography, small-angle X-ray scattering (SAXS), cryo-electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM)
Summary
The endoplasmic reticulum (ER) possesses a sophisticated protein quality control system that ensures the appropriate folding and trafficking of newly synthesised proteins. The quality control system is equipped with an elaborate backup mechanism employing a molecular ‘gatekeeper’ that can catalyse reglucosylation against incompletely folded glycoproteins as potential substrates, thereby regenerating monoglucosylated glycoforms for their return to the chaperone-assisted folding process[3,5]. This gatekeeper function is executed by a unique enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT)[6,7,8,9,10]. We characterise the overall structure of UGGT using integrative biophysical approaches and present the dynamic arrangement of its modular domains
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