Abstract
Protein glycosylation is a critical protein modification. In biogenic membranes of eukaryotes and archaea, these reactions require activated mannose in the form of the lipid conjugate dolichylphosphate mannose (Dol-P-Man). The membrane protein dolichylphosphate mannose synthase (DPMS) catalyzes the reaction whereby mannose is transferred from GDP-mannose to the dolichol carrier Dol-P, to yield Dol-P-Man. Failure to produce or utilize Dol-P-Man compromises organism viability, and in humans, several mutations in the human dpm1 gene lead to congenital disorders of glycosylation (CDG). Here, we report three high-resolution crystal structures of archaeal DPMS from Pyrococcus furiosus, in complex with nucleotide, donor, and glycolipid product. The structures offer snapshots along the catalytic cycle, and reveal how lipid binding couples to movements of interface helices, metal binding, and acceptor loop dynamics to control critical events leading to Dol-P-Man synthesis. The structures also rationalize the loss of dolichylphosphate mannose synthase function in dpm1-associated CDG.
Highlights
Protein glycosylation is a critical protein modification
The overall structure of PfDPMS features an N-terminal, cytoplasmic catalytic domain covalently attached to a TM domain with four TM helices (TMHs) arranged as two TMH dimers, TMD1 and TMD2 (Fig. 1)
The type-I enzymes are represented by dolichylphosphate mannose synthase (DPMS) from Saccharomyces cerevisiae[9], where the catalytic domain is covalently attached to a single C-terminal TMH (Supplementary Fig. 1a)
Summary
In biogenic membranes of eukaryotes and archaea, these reactions require activated mannose in the form of the lipid conjugate dolichylphosphate mannose (Dol-P-Man). We report three highresolution crystal structures of archaeal DPMS from Pyrococcus furiosus, in complex with nucleotide, donor, and glycolipid product. The structures offer snapshots along the catalytic cycle, and reveal how lipid binding couples to movements of interface helices, metal binding, and acceptor loop dynamics to control critical events leading to Dol-P-Man synthesis. In biogenic membranes (i.e., self-synthesizing endoplasmic reticulum (ER) membrane in eukaryotes and cytoplasmic cell membrane in archaea), the activated mannose used for protein glycosylation and glycosylphosphatidylinositol anchor synthesis[1,2,3,4,5,6,7,8] is provided in the form of the glycolipid intermediate dolichylphosphate β-Dmannose (Dol-P-Man). Considering the importance of DPMS function for organism biology, including several common pathogens, these results provide a valuable structural platform for design of therapeutics that target Dol-P-Man-dependent protein glycosylation
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