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

CtUGGT_Kif 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 CtUGGT_Kif 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 CtUGGT_Kif 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 CtUGGT_Kif 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.