期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 289, 期 50, 页码 34900-34910出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.586362
关键词
Computer Modeling; Drug Discovery; Glycosylation; Glycosylation Inhibitor; Ligand-binding Protein; 1; 6-Bisphosphate; PELE; Phosphomannomutase
资金
- Telethon-Italy [GGP12108]
- European Research Council [ERC-2009-Adg 25027-PELE]
- ICREA Funding Source: Custom
Background: Mutations in phosphomannomutase2 cause glycosylation disorder, a disease without a cure that will largely benefit from accurate ligand-bound models. Results: We obtained two models of phospomannomutase2 bound to glucose 1,6-bisphosphate and validated them with limited proteolysis. Conclusion: Ligand binding induces a large conformational transition in PMM2. Significance: We produce and validate closed-form models of PMM2 that represent a starting point for rational drug discovery. The most common glycosylation disorder is caused by mutations in the gene encoding phosphomannomutase2, producing a disease still without a cure. Phosphomannomutase2, a homodimer in which each chain is composed of two domains, requires a bisphosphate sugar (either mannose or glucose) as activator, opening a possible drug design path for therapeutic purposes. The crystal structure of human phosphomannomutase2, however, lacks bound substrate and a key active site loop. To speed up drug discovery, we present here the first structural model of a bisphosphate substrate bound to human phosphomannomutase2. Taking advantage of recent developments in all-atom simulation techniques in combination with limited and site-directed proteolysis, we demonstrated that -glucose 1,6-bisphosphate can adopt two low energy orientations as required for catalysis. Upon ligand binding, the two domains come close, making the protein more compact, in analogy to the enzyme in the crystals from Leishmania mexicana. Moreover, proteolysis was also carried out on two common mutants, R141H and F119L. It was an unexpected finding that the mutant most frequently found in patients, R141H, although inactive, does bind -glucose 1,6-bisphosphate and changes conformation.
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