Using cryo-EM to map small ligands on dynamic metabolic enzymes: Studies with glutamate dehydrogenase

Mario J. Borgnia, Soojay Banerjee, Alan Merk, Doreen Matthies, Alberto Bartesaghi, Prashant Rao, Jason Pierson, Lesley A. Earl, Veronica Falconieri, Sriram Subramaniam, Jacqueline L S Milne

Research output: Contribution to journalArticlepeer-review

Abstract

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.

Original languageEnglish (US)
Pages (from-to)645-651
Number of pages7
JournalMolecular Pharmacology
Volume89
Issue number6
DOIs
StatePublished - Jun 1 2016
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology
  • Molecular Medicine

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