TY - JOUR
T1 - Using cryo-EM to map small ligands on dynamic metabolic enzymes
T2 - Studies with glutamate dehydrogenase
AU - Borgnia, Mario J.
AU - Banerjee, Soojay
AU - Merk, Alan
AU - Matthies, Doreen
AU - Bartesaghi, Alberto
AU - Rao, Prashant
AU - Pierson, Jason
AU - Earl, Lesley A.
AU - Falconieri, Veronica
AU - Subramaniam, Sriram
AU - Milne, Jacqueline L S
PY - 2016/6/1
Y1 - 2016/6/1
N2 - 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.
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.
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U2 - 10.1124/mol.116.103382
DO - 10.1124/mol.116.103382
M3 - Article
C2 - 27036132
AN - SCOPUS:84974560575
SN - 0026-895X
VL - 89
SP - 645
EP - 651
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 6
ER -