Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase

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

doi:10.1016/j.str.2020.11.017

Cited by 18 publications

(14 citation statements)

References 61 publications

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“…We set out to search for ligands of UGGT by fragment-based lead discovery (FBLD) using X-ray crystallography (21)(22)(23)(24). No crystal structures of mammalian UGGTs have been obtained so far, but atomic resolution structures of UGGTs from thermophilic fungi have been determined (25)(26)(27).…”

Section: R a F Tmentioning

confidence: 99%

“…CtUGGT GT24 and U2F CtUGGT GT24 crystal structures FBLD by X-ray crystallography requires the growth of hundreds of well-diffracting crystals of the target macromolecule. None of the crystals of full-length UGGT we grew so far diffracted past 2.8 Å (25,27), but 1.35 and 1.4 Å crystal structures of the catalytic domain of Thermomyces dupontii UGGT (TdUGGT), in complex with UDP and UDP-Glc, respectively, have been described (26).…”

Section: R a F Tmentioning

confidence: 99%

“…FBLD by X-ray crystallography requires the growth of hundreds of well-diffracting crystals of the target macromolecule. None of the crystals of full-length UGGT we grew so far diffracted past 2.8 Å(25, 27), but 1.35 and 1.4 Å crystal structures of the catalytic domain of Thermomyces dupontii UGGT ( Td UGGT), in complex with UDP and UDP-Glc, respectively, have been described(26). Inspired by those results, we cloned the catalytic domain of Ct UGGT without its C-terminal ER-retrieval motif (hereinafter Ct UGGT GT24 ) in the pHLsec vector for secreted mammalian expression(28).…”

Section: Ct Uggtgt24 and U2fctuggtgt24 Crystal Structuresmentioning

confidence: 99%

“…We set out to search for ligands of UGGT by fragment-based lead discovery (FBLD) using X-ray crystallography(21–24). No crystal structures of mammalian UGGTs have been obtained so far, but atomic resolution structures of UGGTs from thermophilic fungi have been determined(25–27). Although compounds binding the N-terminal, misfold-recognising portion of the enzyme would also be potential UGGT inhibitors, we decided to target the C-terminal catalytic domain, given the high 70% similarity and 60% identity between human and fungal sequences in this portion of the enzyme.…”

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confidence: 99%

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A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

Guay¹,

Ibba

Kiappes

et al. 2022

Preprint

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The Endoplasmic Reticulum (ER) glycoprotein folding Quality Control (ERQC) machinery aids folding of glycoproteins in the ER. Misfolded glycoprotein recognition and ER-retention is mediated by the ERQC checkpoint enzyme, the 170 kDa UDP-Glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. Towards the generation of selective UGGT inhibitors, we determined the crystal structures of the catalytic domain of Chaetomium thermophilum UGGT (CtUGGTGT24), alone and in complex with the inhibitor UDP-2-deoxy-2-fluoro-D-glucose (U2F). Using the CtUGGTGT24 crystals, we carried out a fragment-based lead discovery screen via X-ray crystallography and discovered that the small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 'WY' conserved surface motif that is not present in other GT24 family glycosyltransferases. The 5M-8OH-Q molecule has a 613 μM binding affinity for human UGGT1in vitro as measured by saturation transfer difference NMR spectroscopy. The 5M-8OH-Q molecule inhibits both human UGGT1 and UGGT2 activity at concentrations higher than 750 μM in modified HEK293-6E cells. The compound is toxic in cellula and in planta at concentrations higher than 1 mM. A few off-target effects are also observed upon 5M-8OH-Q treatment. Based on an in silico model of the interaction between UGGT and its substrate N-glycan, the 5M-8OH-Q molecule likely works as a competitive inhibitor, binding to the site of recognition of the first GlcNAc residue of the substrate N-glycan.

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Section: R a F Tmentioning

confidence: 99%

Section: R a F Tmentioning

confidence: 99%

Section: Ct Uggtgt24 and U2fctuggtgt24 Crystal Structuresmentioning

confidence: 99%

mentioning

confidence: 99%

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A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

Guay¹,

Ibba

Kiappes

et al. 2022

Preprint

Self Cite

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“…Splitting the additive mixes into two different sets reduced the number of possible cross reactions, but some combinations of mixes were still prone to instability. The 'lanthanides' mix from MORPHEUS II, despite producing some exclusive crystal hits (De Munck et al, 2021;Modenutti et al, 2021), had to be excluded during preliminary tests because it led to precipitation or flocculation when combined with other mixes.…”

Section: Morpheus Additive Mixes Not Integrated Intomentioning

confidence: 99%

The FUSION protein crystallization screen

Gorrec¹,

Bellini²

2022

J Appl Cryst

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The success and speed of atomic structure determination of biological macromolecules by X-ray crystallography depends critically on the availability of diffraction-quality crystals. However, the process of screening crystallization conditions often consumes large amounts of sample and time. An innovative protein crystallization screen formulation called FUSION has been developed to help with the production of useful crystals. The concept behind the formulation of FUSION was to combine the most efficient components from the three MORPHEUS screens into a single screen using a systematic approach. The resulting formulation integrates 96 unique combinations of crystallization additives. Most of these additives are small molecules and ions frequently found in crystal structures of the Protein Data Bank (PDB), where they bind proteins and complexes. The efficiency of FUSION is demonstrated by obtaining high yields of diffraction-quality crystals for seven different test proteins. In the process, two crystal forms not currently in the PDB for the proteins α-amylase and avidin were discovered.

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Analysis of Selenoprotein F Binding to UDP‐Glucose:Glycoprotein Glucosyltransferase (UGGT) by a Photoreactive Crosslinker

et al. 2022

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In the endoplasmic reticulum glycoprotein quality control system, UDP-glucose : glycoprotein glucosyltransferase (UGGT) functions as a folding sensor. Although it is known to form a heterodimer with selenoprotein F (SelenoF), the details of the complex formation remain obscure. A pulldown assay using cotransfected SelenoF and truncated mutants of human UGGT1 (HUGT1) revealed that SelenoF binds to the TRXL2 domain of HUGT1. Additionally, a newly developed photoaffinity crosslinker was selectively introduced into cysteine residues of recombinant SelenoF to determine the spatial orientation of SelenoF to HUGT1. The crosslinking experiments showed that SelenoF formed a covalent bond with amino acids in the TRXL3 region and the interdomain between βS2 and GT24 of HUGT1 via the synthetic crosslinker. SelenoF might play a role in assessing and refining the disulfide bonds of misfolded glycoproteins in the hydrophobic cavity of HUGT1 as it binds to the highly flexible region of HUGT1 to reach its long hydrophobic cavity. Clarification of the SelenoF-binding domain of UGGT and its relative position will help predict and reveal the function of SelenoF from a structural perspective.

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Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase

Cited by 18 publications

References 61 publications

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

The FUSION protein crystallization screen

Analysis of Selenoprotein F Binding to UDP‐Glucose:Glycoprotein Glucosyltransferase (UGGT) by a Photoreactive Crosslinker

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