| Protein is one of the most important biomolecules performing biological functions in the molecular level. Therefore, revealing the microscopic mechanisms of the protein functioning is crucial for understanding the life processes, which becomes one of the major focuses in the interdisciplinary field of physics and biology in recent years.The proteins perform their biological functions mainly through multiscale con-formational fluctuations and interactions. For example, in the catalytic processes of enzymes, the local interaction between the substrate and proteins often induces large scale conformational motions of proteins, which in turn leads to catalytic competent conformations. Such tight coupling between local interactions and global conforma-tional motions is often called as allostery, which is the general functioning mechanism widely used by a number of enzymes, molecular motors, and other protein machines. Adenylate kinase (ADK) is a typical enzyme machine, which is composed of three do-mains, namely, LID domain, CORE domain, and NMP domain, and has been widely used as a model system for studying the molecular mechanisms of folding of multi-domain proteins and allosteric functioning motions of protein machines. The function of ADK is catalyzing the reversible reaction, which maintains the concentration bal-ance between ATP and ADP in cell. Without substrate binding, the ADK adopts an open conformation. Upon the binding of substrate, the ADK switches to closed con-formation. After the catalytic reaction, the substrate dissociates from the binding site, which is coupled with the opening of the LID and NMP domains. Experimental data suggested that the substrate releasing coupled with domain opening is the rate-limiting step of ADK catalytic cycle. However, how does the catalytic product dissociate from the active site and how the product releasing is coupled with the protein conformational motions are largely unclear. Undoubtedly, answering the above questions is the key to understanding the high efficiency of the ADK catalysis. In this dissertation, by using molecular dynamics simulations, we conducted the following three studies:1.Based on metadynamics molecular simulations, we simulated the substrate re-leasing and the coupled conformational motions of adenylate kinase in E-coli (AKE), and constructed the corresponding free energy landscape. Our results showed that the substrate dissociation encounters a relatively high free energy barrier, which is con-sistent with the experimental observation that substrate releasing is the rate-limiting step of the catalytic cycle of ADK. By comparing the results of simulations in which the substrate ADP molecules are protonated with different extent, we found that the protonation of the ADP molecules of the NMP domain can drastically reduce the free energy barrier height, therefore speed up the substrate releasing. We also compared the structure features of the transition state ensemble for the simulations with different protonation states, and revealed the key residues contributing to the substrate releasing process. In addition, our simulations suggested a substrate dissociation pathway, in which the ADP of the LID domain departs from the binding site earlier than that of the ADP of the NMP domain. Particularly, we showed that the substrate releasing is tightly coupled with the protein conformational motions. The substrate releasing requires the nearly full opening of the LID domain and the partial opening of the NMP domain. These results are important for understanding the key dynamics and interaction factors contributing to the substrate releasing and ADK catalytic cycle.2.During the catalytic cycle of ADK, the Mg2+ not only participates in the chem-ical step but also participates in the substrate releasing coupled with conformational motions. It was shown experimentally that one Mg2+ ion coordinates to both ADP molecules right after the chemical step of the catalytic reaction. During the substrate releasing and separation, the Mg2+ may transfer to one of the ADP molecules. How-ever, it is unclear which ADP molecule binds with the Mg2+ during the substrate re-leasing. Such information is critical for understanding the factors controlling the sub-strate releasing and protein conformational opening, which is the rate limit step of the catalysis reaction. In this work, by using metadynamics method, we conducted molec-ular simulations on the adenylate kinase complexed with two ADP molecules and one Mg2+, which corresponds to the post-catalysis enzyme-substrate complex. With such metadynamics simulations, we constructed the free energy landscapes characterizing the Mg2+ transfer to the individual ADP molecules. Our results showed that the Mg2+ has preference to attach with the ADP molecule of the LID domain. Only when the LID domain ADP is protonated, and simultaneously the NMP domain ADP is depro-tonated, the Mg2+ tends to attach with the NMP domain ADP. We also demonstrated that there is ligand exchange process during the Mg2+ transfer. Particularly, the Mg2+ transfer is accompanied with the dehydration process and water rearrangement around the Mg2+. Such kind of effects of Mg2+ transfer on the structure and dynamics of the hydrogen bond network around the active site during the later stage of the catalytic cycle can affect the substrate releasing and the catalytic efficiency.3.To perform biological functions, protein molecules usually need to fold to a certain three-dimensional structure. As a typical multi-domain protein, ADK is of-ten used a model system to study protein folding problem. In the previous studies of ADK folding, people mostly focuses on the spontaneous folding with substrate bind-ing. However, substrate binding may modulate the folding and stability of the ADK. In this dissertation, by using steered molecular dynamics simulations with implicit sol-vent, we studied the unfolding of the AKE under the mechanical force. By comparing the unfolding processes with and without substrate, we showed that substrate binding can modulate the unfolding process of the AKE. Binding of the substrate tends to sta-bilize the domain interface, therefore affect the unfolding pathways. The above results are helpful for understanding the role of substrate on the folding and stabilization of protein molecules.The studies presented in this dissertation may deepen our understanding to the key interaction and dynamics factors contributing to the catalytic relevant functioning motions and the folding/stability of ADK. The content of this dissertation is arranged as following:In chapter 1, we give a brief in introduction to the proteins, enzyme and substrate, and molecular dynamics simulations. In chapter 2, we present the simulation results of the substrate releasing and the coupled protein conformational motions. In chapter 3, we present the simulation results of the Mg2+ transfer between the two ADP molecules during the later stage of the catalytic cycle. The coupled motions of water molecules around the active site are also discussed. In chapter 4, we discuss the effect of substrate binding on the unfolding dynamics of the AKE under the mechanical force. Chapter 5 gives the summary of this dissertation and some prospections for future studies. |
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