Protein O-fucosyltransferase 1 undergoes interdomain flexibility in solution

This article belongs to the Special Issue New Insights into Protein Glycosylation.Protein O-fucosyltransferase 1 (PoFUT1) is a GT-B fold enzyme that fucosylates proteins containing EGF-like repeats. GT-B glycosyltransferases have shown a remarkable grade of plasticity adopting closed and open conformations as a way of tuning their catalytic cycle, a feature that has not been observed for PoFUT1. Here, we analyzed Caenorhabditis elegans PoFUT1 (CePoFUT1) conformational behavior in solution by atomic force microscopy (AFM) and single-molecule fluorescence resonance energy transfer (SMF-FRET). Our results show that this enzyme is very flexible and adopts mainly compact conformations and to a lesser extend a highly dynamic population that oscillates between compact and highly extended conformations. Overall, our experiments illustrate the inherent complexity of CePoFUT1 dynamics, which might play a role during its catalytic cycle.We thank ARAID: MEC (CTQ2013-44367-C2-2-P, BFU2016-75633-P and PID2019-105451GB-I00 to RH-G, CTQ2017-85658-R and CTQ2014-56370-R to AO), and Gobierno de Aragón (E35_R20 and LMP58_18) with FEDER (2014-2020) funds for ‘Building Europe from Aragón’ for financial support. EL-N acknowledges her postdoctoral Juan de la Cierva fellowship IJCI-2017-32874 supported by the Ministry of Science and Innovation (Spain).Peer reviewe