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Crystal structure determination of CtUGGT from HEK293F cells treated with 5 μM kifunensine.

Pietro Roversi; Thomas Waksman; Dominic S. Alonzi

CtUGGTKif was purified from the supernatant of HEK293F cells treated with 5 mM kifunensine and transfected with the pHLsec:CtUGGT vector for secreted expression of Chaetomium thermophilum UGGT (CtUGGT). The protein carries high-mannose glycans, as evidenced by EndoF1 cleavage. A crystal was grown from the CtUGGTKif sample and diffraction data were collected on I03@Diamond on 01.05.2016. Structure factor amplitudes scaled to 4.1 Å resolution (after anisotropic scaling) and the phases were determined by molecular replacement using PDB ID 5NV4 as a search model. The crystals belong to the same P212121 crystal form already deposited as PDB ID 5NV4 for the CtUGGT D611C:G1050C double mutant. The CtUGGTKif structure (deposited as PDB ID 6TRF) combines the close inter-domain distance between the TRXL2 and βS2 domains observed in the CtUGGT "closed-like conformation" (PDB ID 5NV4) with the larger inter-domain distance between the TRXL1 and TRXL3 domains observed in the CtUGGT "open conformation" (PDB ID 5MZO). This CtUGGTKif crystal structure suggests that the UGGT molecule can close the gap between the TRXL2 and βS2 domains while at the same time opening the one between the TRXL1 and TRXL3 domains. The disorder observed in the TRXL2 domain in the crystal may be a clue that UGGT bears an intrinsically disordered domain, a finding that would explain how UGGT can recognise misfolded glycoproteins.

Cloning, protein expression, purification and crystallisation took place at the Department of Biochemistry, Oxford University and were supported and funded by Prof. Nicole Zitzmann through the Oxford Glycobiology Institute Endowment. Thanks to Ed Lowe for the management of the Crystallography Facility and the coordination of the diffraction experiments. PR is funded by a LISCB/Wellcome Trust ISSF Fellowship 204801/Z/16/Z, and a UK Wellcome Trust Seed Award in Science 214090/Z/18/Z . Thomas Waksman was a Biochemistry MSc student at the Biochemistry Dept. of Oxford University and St. Edmund's Hall College, Oxford, England, UK.
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