 Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant PathologiesBenedikt Frieg, Boris Görg, Nadine Homeyer, Verena Keitel, Dieter Häussinger and Holger Gohlke PLOS Computational Biology, 2016, vol. 12, issue 2, 1-28 Abstract: Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.Author Summary: Glutamine synthetase (GS) catalyzes the ATP-dependent ligation of ammonia and glutamate to glutamine, which makes the enzyme essential for human nitrogen metabolism. Three mutations in human GS, R324C, R324S, and R341C, had been identified previously that lead to a glutamine deficiency, resulting in neonatal death in the case of R324C and R341C. However, the molecular mechanisms underlying this impairment of GS activity have remained elusive. Our results from computational biophysics approaches suggest that all three mutants influence the first step of GS’ catalytic cycle, namely ATP or glutamate binding. The analyses reveal a complex set of effects including the loss of direct interactions to substrates, the involvement of water-mediated interactions that alleviate part of the mutation effect, and long-range effects between the catalytic site and structural parts distant from it. As to the latter, experimental validation is in line with our prediction of a significant destabilization of helix H8 in the R341C mutant, which should negatively affect glutamate binding. Finally, our findings could stimulate the development of ATP-binding enhancing molecules for the R324S mutant, which has been suggested to have residual activity, that way extrinsically “repairing” the mutant. Date: 2016 Downloads: (external link) Related works: Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text Persistent link: https://EconPapers.repec.org/RePEc:plo:pcbi00:1004693 DOI: 10.1371/journal.pcbi.1004693 Access Statistics for this article More articles in PLOS Computational Biology from Public Library of Science |
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