    2.4.1.83 | purified PfDPMS with bound GDP-Man and Mn2+ and with bound GDP and Mg2+, and acceptor substrate Dol55-P, sitting drop vapor diffusion method, 15-20 mg/ml protein is mixed with 5 mM MgCl2 or MnCl2 and either 5 mM GDP or GDP-Man in 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% v/v glycerol, and 0.05% LDAO, the protein solution is mixed with crystallization solution containing 0.2 M potassium chloride, 0.1 M trisodium citrate, pH 5.5, and 37% v/v pentaerythritol propoxylate, 4°C, X-ray crystal structure determination and analysis, Dol55-P-Man structure modeling |
| 2.4.1.83 | structures of DPMS, in complex with nucleotide Mg2+, donor GDP-mannose, and glycolipid product. Substrate binding and product release are orchestrated by an interplay between juxtamembrane interface helices, donor, metal ion and acceptor. Displacement and conformational changes of the juxtamembrane interface helices, most pronouncedly of IFH2, enable entry of the dolichol-phosphate acceptor between IFH1 and IFH2, and docking of its phosphate group at Ser135 (assisted by Arg117 and Arg131), which is located immediately below the mannosyl to be transferred. The acceptor-induced changes in the juxtamembrane interface helices lead to disruption of the A-loop interaction network at the DXD motif and dislodgement of the A-loop. Collapse of the interaction network and opening of the A-loop coincides with concomitant loss of metal ion and attack by the pre-activated nucleophilic dolichol phosphate oxygen on the mannosyl C1 atom |
