TY - JOUR
T1 - Energy landscape of domain motion in glutamate dehydrogenase deduced from cryo-electron microscopy
AU - Oide, Mao
AU - Kato, Takayuki
AU - Oroguchi, Tomotaka
AU - Nakasako, Masayoshi
N1 - Funding Information:
The authors thank Professor Keiichi Namba of Osaka University for the use of the EM facility. Trp89Phe‐mutated GDH was purified and crystallized by Ms. Yuka Matsui. This study was supported by grants from the Japan Society for the Promotion of Science (Nos. jp13480214, jp19204042, and jp22244054 to M.N.; Nos. jp26800227 and jp17H04854 to TO.; and No. 18J11653 to MO) and by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Nos. jp15076210, jp20050030, jp22018027, jp23120525, jp25120725, jp15H01647, and jp17H05891 to MN; and No. jp26104535 to TO). CryoEM observations were partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research [Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)] from the Japan Agency for Medical Research and Development (AMED) (project No. 0436). X‐ray diffraction data collection of mutated GDH crystal was performed at BL26B2 with the approval of the RIKEN Harima Institute.
Publisher Copyright:
© 2020 Federation of European Biochemical Societies
PY - 2020/8/1
Y1 - 2020/8/1
N2 - Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of unliganded glutamate dehydrogenase from Thermococcus profundus using cryo-electron microscopy and proposed a novel method to construct free-energy landscape of protein conformations. Each subunit of the homo-hexameric enzyme comprises nucleotide-binding domain (NAD domain) and hexamer-forming core domain. A large active-site cleft is situated between the two domains and varies from open to close according to the motion of a NAD domain. A three-dimensional map reconstructed from all cryo-electron microscopy images displayed disordered volumes of NAD domains, suggesting that NAD domains in the collected images adopted various conformations in domain motion. Focused classifications on NAD domain of subunits provided several maps of possible conformations in domain motion. To deduce what kinds of conformations appeared in EM images, we developed a novel analysis method that describe the EM maps as a linear combination of representative conformations appearing in a 200-ns molecular dynamics simulation as reference. The analysis enabled us to estimate the appearance frequencies of the representative conformations, which illustrated a free-energy landscape in domain motion. In the open/close domain motion, two free-energy basins hindered the direct transformation from open to closed state. Structure models constructed for representative EM maps in classifications demonstrated the correlation between the energy landscape and conformations in domain motion. Based on the results, the domain motion in glutamate dehydrogenase and the analysis method to visualize conformational changes and free-energy landscape were discussed. Database: The EM maps of the four conformations were deposited to Electron Microscopy Data Bank (EMDB) as accession codes EMD-9845 (open), EMD-9846 (half-open1), EMD-9847 (half-open2), and EMD-9848 (closed), respectively. In addition, the structural models built for the four conformations were deposited to the Protein Data Bank (PDB) as accession codes 6JN9 (open), 6JNA (half-open1), 6JNC (half-open2), and 6JND (closed), respectively.
AB - Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of unliganded glutamate dehydrogenase from Thermococcus profundus using cryo-electron microscopy and proposed a novel method to construct free-energy landscape of protein conformations. Each subunit of the homo-hexameric enzyme comprises nucleotide-binding domain (NAD domain) and hexamer-forming core domain. A large active-site cleft is situated between the two domains and varies from open to close according to the motion of a NAD domain. A three-dimensional map reconstructed from all cryo-electron microscopy images displayed disordered volumes of NAD domains, suggesting that NAD domains in the collected images adopted various conformations in domain motion. Focused classifications on NAD domain of subunits provided several maps of possible conformations in domain motion. To deduce what kinds of conformations appeared in EM images, we developed a novel analysis method that describe the EM maps as a linear combination of representative conformations appearing in a 200-ns molecular dynamics simulation as reference. The analysis enabled us to estimate the appearance frequencies of the representative conformations, which illustrated a free-energy landscape in domain motion. In the open/close domain motion, two free-energy basins hindered the direct transformation from open to closed state. Structure models constructed for representative EM maps in classifications demonstrated the correlation between the energy landscape and conformations in domain motion. Based on the results, the domain motion in glutamate dehydrogenase and the analysis method to visualize conformational changes and free-energy landscape were discussed. Database: The EM maps of the four conformations were deposited to Electron Microscopy Data Bank (EMDB) as accession codes EMD-9845 (open), EMD-9846 (half-open1), EMD-9847 (half-open2), and EMD-9848 (closed), respectively. In addition, the structural models built for the four conformations were deposited to the Protein Data Bank (PDB) as accession codes 6JN9 (open), 6JNA (half-open1), 6JNC (half-open2), and 6JND (closed), respectively.
KW - cryo-electron microscopy
KW - domain motion
KW - energy landscape
KW - glutamate dehydrogenase
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U2 - 10.1111/febs.15224
DO - 10.1111/febs.15224
M3 - Article
C2 - 31976609
AN - SCOPUS:85078897323
VL - 287
SP - 3472
EP - 3493
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 16
ER -