PT  - JOURNAL ARTICLE
AU  - Mario J. Borgnia
AU  - Soojay Banerjee
AU  - Alan Merk
AU  - Doreen Matthies
AU  - Alberto Bartesaghi
AU  - Prashant Rao
AU  - Jason Pierson
AU  - Lesley A. Earl
AU  - Veronica Falconieri
AU  - Sriram Subramaniam
AU  - Jacqueline L. S. Milne
TI  - Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase
AID  - 10.1124/mol.116.103382
DP  - 2016 Jun 01
TA  - Molecular Pharmacology
PG  - 645--651
VI  - 89
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/89/6/645.short
4100  - http://molpharm.aspetjournals.org/content/89/6/645.full
SO  - Mol Pharmacol2016 Jun 01; 89
AB  - 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.