Abstract
SummaryUDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name “Parodi limit” the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70–80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a “one-size-fits-all adjustable spanner” UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.
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
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)
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