Structural basis for dolichylphosphate mannose biosynthesis

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.The generation of glycolipid dolichylphosphate mannose (Dol-P-Man) is a critical step for protein glycosylation and GPI anchor synthesis. Here the authors report the structure of dolichylphosphate mannose synthase in complex with bound nucleotide and donor to provide insight into the mechanism of Dol-P-Man synthesis.

Fig. 1

Overall structure of PfDPMS. a Structure of PfDPMS with bound GDP-Man and Mn²⁺. The acceptor loop (purple) is locked in a closed conformation. b Two-fold pseudo symmetry of the TMD1/TMD2 interface. c Sequence similarity between TMD1 and TMD2, including the residues that define the pseudo twofold symmetry in b

Fig. 2

Details of nucleotide and donor binding. a The PfDPMS active site with bound GDP•Mg²⁺ and b GDP-Man•Mn²⁺ in the absence of acceptor substrate. The acceptor loop (purple) is locked in a closed conformation by ionic bonds between Glu12 and Arg202 and Lys208 close to the GDP α-phosphate. The IF helices do not participate directly in donor binding. Asp39 interacts with the N1 and N2 atoms of the guanine base, and Phe177 acts as a gate and closes off the pocket exit. c Superimposition of the PfDPMS•GDP•Mg²⁺ (beige) and PfDPMS•GDP-Man•Mn²⁺ (light blue) complexes

Fig. 3

Structure of the PfDPMS•GDP•Dol55-P-Man complex. a Enlarged views of the active site (left inset) and the TM domain (right inset). Left inset, the Dol-P-Man head group is shown bound in the active site of wild-type PfDPMS. The metal ion has departed and the GDP diphosphate group has flipped away from the transfer site towards the “back door”, to accommodate the glycolipid product. Right inset, the Dol-P-Man isoprenoid chain traces down to the TM domain where it rests on TMH4. The acceptor loop is open and Phe177 is in the “down” position. Superimposition of the crystal complexes b PfDPMS•GDP•Mg²⁺ (gold) and PfDPMS•GDP•Dol55-P-Man (blue), and c PfDPMS•GDP-Man•Mn²⁺ (gold) and PfDPMS•GDP•Dol55-P-Man (blue). Relevant side chains are shown

Fig. 4

Catalytic mechanism of PfDPMS. Catalytic mechanism of the transfer reaction. Nucleophilic attack by the Dol-P phosphate oxygen on the mannosyl C1 carbon yields GDP and Dol-P-Man. Asp91 and Gln93 coordinate the GDP-Man diphosphate groups via the metal ion, while the key side chains for positioning the Dol-P phosphate group for mannosyl transfer are Ser135 and Arg117

Fig. 5

Activity of PfDPMS wild type and truncation variant Δ230–352. Amount of product formed measured as nanomoles of released free phosphate for wild-type PfDPMS and the truncation variant Δ230–352 lacking the TM domain. Dol55-P was used as acceptor substrate. Δ230–352 _m and Δ230–352 _s were purified from the membrane fraction and aqueous phase, respectively; and they display catalytic activity comparable with that of the wild type. Errors are given as mean values ± s.e.m. (N = 3)

Fig. 6

Proposed model of substrate binding and product release. Schematic representation of the conformational changes associated with substrate binding and product release. Step 1: prior to encounter with a Dol-P acceptor molecule. GDP-Man and metal ion are bound in the active site. Step 2: Dol-P binding, IFH2 movement, opening of “back door” and of the acceptor loop. Step 3: glycosyl transfer followed and subsequent release of GDP and metal ion. Step 4: binding of GDP-Man and metal ion, release of Dol-P-Man, closure of back door and acceptor loop. See text for further details. Color scheme: Catalytic domain, blue; IFH2, green; TM domain, red; acceptor loop, pink; GDP-Man, orange and yellow ovals; metal ion, purple circle; Phe177, gray oval; Dol-P isoprenoid chain, gray; and Dol-P phosphate group, green circle