RT Journal Article
SR Electronic
T1 Using cryo-EM to map small ligands on dynamic metabolic enzymes: Studies with glutamate dehydrogenase
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.116.103382
DO 10.1124/mol.116.103382
A1 Mario J. Borgnia
A1 Soojay Banerjee
A1 Alan Merk
A1 Doreen Matthies
A1 Alberto Bartesaghi
A1 Prashant Rao
A1 Jason Pierson
A1 Lesley A. Earl
A1 Veronica Falconieri
A1 Sriram Subramaniam
A1 Jacqueline L. S. Milne
YR 2016
UL http://molpharm.aspetjournals.org/content/early/2016/04/04/mol.116.103382.abstract
AB Cryo-EM methods are rapidly 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 330 kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurological 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. While 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 where 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.