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A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation.

Lu J, Jiang C, Li X, Jiang L, Li Z, Schneider-Poetsch T, Liu J, Yu K, Liu JO, Jiang H, Luo C, Dang Y - Nucleic Acids Res. (2015)

Bottom Line: Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation.Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI.Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Metabolism and Molecular Medicine, the Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

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Simulation of Pi release using LES approach. (A) The probability of Pi release from wild type eIF4AI (WT) and eIF4AI with SAT/AAA mutation (SAT/AAA) at different temperatures. Simulations were starting from the conformations in which the channel was open or closed. All parts of the system were allowed to move during the simulations. The error bars represent the standard error of mean (SEM). (B) The Pi release pathway is shown as a mesh surface enclosing the space that highly occupied by the phosphorus atom (occupancy rate > 0.1) in the trajectories of Pi successfully escaping from hydrolysis site (calculated by VolMap in VMD). Residues around the Pi release channel are shown as sticks. (C) Box plot showing the distribution of RMSD values of the backbone atoms of the linker and the whole protein and heavy atoms of RNA in LES trajectories with or without Pi release. The RMSD value was calculated between structure from the final frame in each trajectory and the starting structure. LES trajectory was defined as ‘Pi released’ trajectory when at least one Pi replicate escaped from the hydrolysis site. (D) Pi release probability from wild type eIF4AI and several mutants at 500 K. The error bars represent SEM.
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Figure 3: Simulation of Pi release using LES approach. (A) The probability of Pi release from wild type eIF4AI (WT) and eIF4AI with SAT/AAA mutation (SAT/AAA) at different temperatures. Simulations were starting from the conformations in which the channel was open or closed. All parts of the system were allowed to move during the simulations. The error bars represent the standard error of mean (SEM). (B) The Pi release pathway is shown as a mesh surface enclosing the space that highly occupied by the phosphorus atom (occupancy rate > 0.1) in the trajectories of Pi successfully escaping from hydrolysis site (calculated by VolMap in VMD). Residues around the Pi release channel are shown as sticks. (C) Box plot showing the distribution of RMSD values of the backbone atoms of the linker and the whole protein and heavy atoms of RNA in LES trajectories with or without Pi release. The RMSD value was calculated between structure from the final frame in each trajectory and the starting structure. LES trajectory was defined as ‘Pi released’ trajectory when at least one Pi replicate escaped from the hydrolysis site. (D) Pi release probability from wild type eIF4AI and several mutants at 500 K. The error bars represent SEM.

Mentions: To test whether the opening of the backdoor channel, which results from the conformational change of the inter-domain linker, is related to Pi release, we simulated the Pi release form eIF4AI conformation with and without the existence of the backdoor channel, which represents the ‘ready to release’ and ‘Pi withholding’ states, respectively. As the timescale of the actual Pi release is on the order of milli-seconds or longer, which is beyond the time scale of cMD simulation, we adopted an approach similar to the one used by Martin Karplus et al. in simulating Pi release from myosin (21). The implementation of this approach in the Amber package is called locally enhanced sampling (LES) (33). In LES, the ligands (Pi in the present study) can be kept at a high temperature to speed up the crossover of energy barriers while the protein is kept at 300 K to avoid unrealistic motions (41). The detailed simulation procedure is described in Materials and Methods. Figure 3A shows the probabilities of Pi release from eIF4AI as a function of temperature. In the channel open state, Pi molecules started to be released at 500 K and 90% of Pi molecules escaped from the hydrolysis site at 700 K (red line). In contrast, only a small fraction of Pi molecules (∼30%) escaped from the hydrolysis site at 700 K (black line) in the channel closed state. By calculating the space occupancy of Pi molecules in the trajectories that all Pi replicates escaped from the hydrolysis site, we found the Pi release pathways observed in the LES simulations starting from the ‘channel open’ state or the ‘channel closed’ state overlapped with the backdoor channel observed in the cMD simulation (Figure 3B, Supplementary Movie S2). In addition, through analyzing protein structures in LES trajectories with and without Pi release, we found Pi release was accompanied by further movement of the linker while the overall protein and RNA structure remained stable (Figure 3C). From the movie that shows a typical Pi release from eIF4AI in the channel closed state, a significant conformational change of the linker can be observed before Pi escaping (Supplementary Movie S2). Therefore, these results suggest that the conformational change of the linker and opening of the backdoor channel indeed promote Pi release from eIF4AI in the ADP-Pi bound state.


A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation.

Lu J, Jiang C, Li X, Jiang L, Li Z, Schneider-Poetsch T, Liu J, Yu K, Liu JO, Jiang H, Luo C, Dang Y - Nucleic Acids Res. (2015)

Simulation of Pi release using LES approach. (A) The probability of Pi release from wild type eIF4AI (WT) and eIF4AI with SAT/AAA mutation (SAT/AAA) at different temperatures. Simulations were starting from the conformations in which the channel was open or closed. All parts of the system were allowed to move during the simulations. The error bars represent the standard error of mean (SEM). (B) The Pi release pathway is shown as a mesh surface enclosing the space that highly occupied by the phosphorus atom (occupancy rate > 0.1) in the trajectories of Pi successfully escaping from hydrolysis site (calculated by VolMap in VMD). Residues around the Pi release channel are shown as sticks. (C) Box plot showing the distribution of RMSD values of the backbone atoms of the linker and the whole protein and heavy atoms of RNA in LES trajectories with or without Pi release. The RMSD value was calculated between structure from the final frame in each trajectory and the starting structure. LES trajectory was defined as ‘Pi released’ trajectory when at least one Pi replicate escaped from the hydrolysis site. (D) Pi release probability from wild type eIF4AI and several mutants at 500 K. The error bars represent SEM.
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Related In: Results  -  Collection

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Figure 3: Simulation of Pi release using LES approach. (A) The probability of Pi release from wild type eIF4AI (WT) and eIF4AI with SAT/AAA mutation (SAT/AAA) at different temperatures. Simulations were starting from the conformations in which the channel was open or closed. All parts of the system were allowed to move during the simulations. The error bars represent the standard error of mean (SEM). (B) The Pi release pathway is shown as a mesh surface enclosing the space that highly occupied by the phosphorus atom (occupancy rate > 0.1) in the trajectories of Pi successfully escaping from hydrolysis site (calculated by VolMap in VMD). Residues around the Pi release channel are shown as sticks. (C) Box plot showing the distribution of RMSD values of the backbone atoms of the linker and the whole protein and heavy atoms of RNA in LES trajectories with or without Pi release. The RMSD value was calculated between structure from the final frame in each trajectory and the starting structure. LES trajectory was defined as ‘Pi released’ trajectory when at least one Pi replicate escaped from the hydrolysis site. (D) Pi release probability from wild type eIF4AI and several mutants at 500 K. The error bars represent SEM.
Mentions: To test whether the opening of the backdoor channel, which results from the conformational change of the inter-domain linker, is related to Pi release, we simulated the Pi release form eIF4AI conformation with and without the existence of the backdoor channel, which represents the ‘ready to release’ and ‘Pi withholding’ states, respectively. As the timescale of the actual Pi release is on the order of milli-seconds or longer, which is beyond the time scale of cMD simulation, we adopted an approach similar to the one used by Martin Karplus et al. in simulating Pi release from myosin (21). The implementation of this approach in the Amber package is called locally enhanced sampling (LES) (33). In LES, the ligands (Pi in the present study) can be kept at a high temperature to speed up the crossover of energy barriers while the protein is kept at 300 K to avoid unrealistic motions (41). The detailed simulation procedure is described in Materials and Methods. Figure 3A shows the probabilities of Pi release from eIF4AI as a function of temperature. In the channel open state, Pi molecules started to be released at 500 K and 90% of Pi molecules escaped from the hydrolysis site at 700 K (red line). In contrast, only a small fraction of Pi molecules (∼30%) escaped from the hydrolysis site at 700 K (black line) in the channel closed state. By calculating the space occupancy of Pi molecules in the trajectories that all Pi replicates escaped from the hydrolysis site, we found the Pi release pathways observed in the LES simulations starting from the ‘channel open’ state or the ‘channel closed’ state overlapped with the backdoor channel observed in the cMD simulation (Figure 3B, Supplementary Movie S2). In addition, through analyzing protein structures in LES trajectories with and without Pi release, we found Pi release was accompanied by further movement of the linker while the overall protein and RNA structure remained stable (Figure 3C). From the movie that shows a typical Pi release from eIF4AI in the channel closed state, a significant conformational change of the linker can be observed before Pi escaping (Supplementary Movie S2). Therefore, these results suggest that the conformational change of the linker and opening of the backdoor channel indeed promote Pi release from eIF4AI in the ADP-Pi bound state.

Bottom Line: Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation.Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI.Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Metabolism and Molecular Medicine, the Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

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