Protein glycosylation is an essential posttranslational modification influencing protein function and cell-cell signaling. This process relies on enzymes that build a conserved glycan, with phosphoglycosyltransferases often performing critical steps. One such enzyme, the homodimer DPAGT1, catalyzes the first, committed step in the human N-linked glycosylation pathway, and its misregulation and mutation are directly linked with myriad cancers and congenital disorders. However, there are no approved treatments for DPAGT1-related diseases, and little is known about its mechanistic details. To address these problems, we pursued cryo-EM studies. DPAGT1 is a challenging target, as it is a sub-100 kDa integral membrane protein. Nevertheless, we were able to obtain sub-3 Å cryo-EM reconstructions of DPAGT1 without the use of fiducial markers, and we present the first cryo-EM structures of DPAGT1 to our knowledge. DPAGT1 with a novel anticancer inhibitor reveals local protein—inhibitor contacts and highlights sites for further optimization in drug design. An apo DPAGT1 structure clarifies lingering questions from previous crystal structures, like the binding position of a catalytically relevant lipid. It also identifies interactions at the dimer interface that were not previously resolved, and these reveal symmetry breaking features, which appear related to the enzyme’s function. Together, these structures inform future efforts to target DPAGT1 therapeutically and provide new insights into phosphoglycosyltransferase mechanisms.
Kirsh et al. (Sun,) studied this question.