CryoEM-sampling of metastable conformations appearing in cofactor-ligand association and catalysis of glutamate dehydrogenase.

Kinetic aspects of enzymatic reactions are described by equations based on the Michaelis-Menten theory for the initial stage. However, the kinetic parameters provide little information on the atomic mechanism of the reaction. In this study, we analyzed structures of glutamate dehydrogenase in the initial and steady stages of the reaction using cryoEM at near-atomic resolution. In the initial stage, four metastable conformations displayed different domain motions and cofactor/ligand association modes. The most striking finding was that the enzyme-cofactor-substrate complex, treated as a single state in the enzyme kinetic theory, comprised at least three different metastable conformations. In the steady stage, seven conformations, including derivatives from the four conformations in the initial stage, made the reaction pathway complicated. Based on the visualized conformations, we discussed stage-dependent pathways to illustrate the dynamics of the enzyme in action.

Coenzyme-binding pathway on glutamate dehydrogenase suggested from multiple-binding sites visualized by cryo-electron microscopy.

The structure of hexameric glutamate dehydrogenase (GDH) in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP) was visualized using cryogenic transmission electron microscopy to investigate the ligand-binding pathways to the active site of the enzyme. Each subunit of GDH comprises one hexamer-forming core domain and one nucleotide-binding domain (NAD domain), which spontaneously opens and closes the active-site cleft situated between the two domains. In the presence of NADP, the potential map of GDH hexamer, assuming D3 symmetry, was determined at a resolution of 2.4 Å, but the NAD domain was blurred due to the conformational variety. After focused classification with respect to the NAD domain, the potential maps interpreted as NADP molecules appeared at five different sites in the active-site cleft. The subunits associated with NADP molecules were close to one of the four metastable conformations in the unliganded state. Three of the five binding sites suggested a pathway of NADP molecules to approach the active-site cleft for initiating the enzymatic reaction. The other two binding modes may rarely appear in the presence of glutamate, as demonstrated by the reaction kinetics. Based on the visualized structures and the results from the enzymatic kinetics, we discussed the binding modes of NADP to GDH in the absence and presence of glutamate.

Assessment of Force Field Accuracy Using Cryogenic Electron Microscopy Data of Hyper-thermostable Glutamate Dehydrogenase.

Molecular dynamics (MD) simulations in biophysically relevant time scales of microseconds is a powerful tool for studying biomolecular processes, but results often display force field dependency. Therefore, assessment of force field accuracy using experimental data of biomolecules in solution is essential for simulation studies. Here, we propose the use of structural models obtained via cryo-electron microscopy (cryoEM), which provides biomolecular structures in vitreous ice mimicking the environment in solution. The accuracy of the AMBER (ff99SB-ILDN-NMR, ff14SB, ff15ipq, and ff15FB) and CHARMM (CHARMM22 and CHARMM36m) force fields was assessed by comparing their MD trajectories with the cryoEM data of thermostable hexameric glutamate dehydrogenase (GDH), which included a cryoEM map at a resolution of approximately 3 Å and structure models of subunits reflecting metastable conformations in domain motion occurring in GDH. In the assessment, we validated the force fields with respect to the reproducibility and stability of secondary structures and intersubunit interactions in the cryoEM data. Furthermore, we evaluated the force fields regarding the reproducibility of the energy landscape in the domain motion expected from the cryoEM data. As a result, among the six force fields, ff15FB and ff99SB-ILDN-NMR displayed good agreement with the experiment. The present study demonstrated the advantages of the high-resolution cryoEM map and suggested the optimal force field to reproduce experimentally observed protein structures.