Clamping, Bending, and Twisting Inter-Domain Motions in the Misfold-Recognising Portion of UDP-Glucose: Glycoprotein Glucosyl-Transferase

Carlos P Modenutti,Snežana Vasiljević,Lucia Marti,Anu V Chandran,Roberta Ibba,Juan I Blanco Capurro,Nicole Zitzmann,Pietro Roversi,Angelo Santino,Mario Hensen,Dominic S Alonzi,Jonathan Rushton,Gábor Tax,Abhinav Kumar,Johan C Hill,Andrea Lia,Marcelo A Martí,Thomas Waksman,Simone Rubichi

doi:10.2139/ssrn.3565029

Abstract

UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the glycoprotein folding checkpoint in the eukaryotic glycoprotein secretory pathway. The enzyme detects misfolded glycoproteins in the Endoplasmic Reticulum, and flags them for retention by re-glucosylating them on an N-linked glycan. The fit of a UGGT crystal structure to a negative stain electron microscopy reconstruction of UGGT in complex with an antibody suggests that the misfold-sensing N-terminal portion of UGGT and its C-terminal catalytic domain are tightly associated. Molecular Dynamics (MD) simulations capture UGGT in so far unobserved conformational states, and principal component analysis of the MD trajectories affords a description of UGGT's overall inter-domain motions, highlighting three types of inter-domain movements: bending, twisting and clamping. These inter-domain motions modify the accessible surface area of the enzyme's central saddle. We propose to name the maximum distance between a site of misfolding on a UGGT glycoprotein substrate and an N-linked glycan that monomeric UGGT can re-glucosylate on the same glycoprotein. MD simulations estimate the Parodi limit to be around 60-70 A. Re-glucosylation assays using UGGT deletion mutants suggest that the TRXL2 domain is necessary for activity against urea-misfolded bovine thyroglobulin. Our findings support a one-size-fits-all adjustable spanner substrate recognition model, with a crucial role for the TRXL2 domain in the recruitment of misfolded substrates.

Highlights

A wonderfully efficient protein-folding machinery in the ER of eukaryotic cells ensures that only correctly folded glycoproteins can exit the ER, proceed to the Golgi, and from there continue along the secretory pathway toward their cellular or extracellular destinations (Vincenz-Donnelly and Hipp, 2017)
We define and give a numerical estimate of the ‘‘Parodi limit,’’ the maximum distance between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UDP-glucose:glycoprotein glucosyltransferase (UGGT) on the same glycoprotein in vitro in response to recognition of misfold at that site
The CtUGGTKif crystal structure adds to the landscape sampled by previously observed UGGT conformations The full-length Chaetomium thermophilum UGGT (CtUGGT) crystal structures revealed four DsbA-like domains (TRXL1–4) arranged in a long arc, terminating in two b sandwiches tightly clasping the glucosyltransferase family 24 (GT24) domain (Figures 1A and 1B) (Roversi et al, 2017)

Summary

Introduction

A wonderfully efficient protein-folding machinery in the ER of eukaryotic cells ensures that only correctly folded glycoproteins can exit the ER, proceed to the Golgi, and from there continue along the secretory pathway toward their cellular or extracellular destinations (Vincenz-Donnelly and Hipp, 2017). ERQC-mediated ER retention and ERAD degradation of glycoprotein mutants bear unfortunate consequences when the mutation induces a minor folding defect but does not abrogate the function of the glycoprotein (‘‘responsive mutant’’). In these cases ERQC/ERAD cause disease by blocking the secretion of the glycoprotein mutant, even though its residual activity would be beneficial to the organism (see for example Parodi et al, 2014)

Methods

Results

Discussion

Conclusion