| Living cells and life processes depend on the three-dimensional(3D)structure of biological macromolecules,which are the essential executors of cellular functions.Thus,biological macromolecular structures are crucial to the understanding of biological processes and play a pivotal role in molecular genetics,enzymology,biochemistry,and diseases.Nuclear Magnetic Resonance(NMR),X-ray crystallography,and cryo-electron microscopy(cryoEM)are three sought-after technologies for macromolecular structural determination.In order to figure out the intrinsic mechanism between structure and function of the resolved proteins,computational biology related methods were often employed to provide abundant structural relevant information.Among these available methods,molecular dynamic simulation stands out with increasing popularity.With the increase of computational power,application of this method was changing gradually from small organic molecules to many crucial proteins,such as enzyme,membrane protein,ribosome,and virus capsid.In this thesis,we studied the relationship between structure and function at varying levels based on three vital protein systems by advanced computational and experimental means.The first system is Ralstonia solanacearum lectin(RSL)-(methyl-?-L-fucoside)binding complex,which is a model system for the studies of carbohydrate-protein association.Carbohydrate-protein associations participate in and regulate a variety of biologically relevant processes such as cell-cell adhesion,cell differentiation,and in-cell signaling.The capability of molecular recognition depends on specific interactions between protein and carbohydrate.A method that can reliably predict protein-carbohydrate binding free energies is a basis for rational drug design for the complexes in this catalog.Enormous efforts have been dedicated to this field.However,it is still challenging for carbohydrates due to their flexibility,which is the case for methyl-?-L-fucoside.In this study,both the general AMBER force field(GAFF)and GLYCAM06j were applied for methyl-?-L-fucoside description.The free energy at the molecular mechanical(MM)level was calculated using the double decoupling method(DDM)and the free energy change at each step was calculated via a series of intermediate states using the Bennett acceptance ratio(BAR).GLYCAM06j is a carbohydrate-specific force field and therefore provides relatively accurate results for binding free energy than GAFF does.The quantum mechanical/molecular mechanical(QM/MM)free energy was calculated by thermodynamic perturbation(TP)from the MM states.Unexpectedly,after this MM-to-QM/MM correction,the agreement with the experimental value decreased.These studies indicate that:(1)During the decoupling process,the binding between RSL and methyl-?-L-fucoside is gradually attenuated.Thus we observed slow convergence for some intermediate states.(2)Consistent distributions are required for six harmonic restraints added to two terminal states along the thermodynamic cycle.(3)Further improvements for QM/MM correction methods are needed,especially for the description of the interaction between QM and MM regions.The study of this important protein-binding system promotes our understanding for carbohydrate-protein binding and serves as a reference for the carbohydrate binding-related studies in the future.The second system is 3-phosphoinositide-dependent protein kinase-1(PDK1)-SBF1 binding complex,which is a selected system for phosphorylation protein-ligand association studies.The study of protein phosphorylation has a pivotal role that underlies the regulation of normal cells,and participates in signaling pathways which can be subverted in human disease.The phosphorylation proteins thus become increasingly crucial.The design of allosteric inhibitor with high affinity,high selectivity,and high specificity also was becoming a hot field of many interests.The overexpression of PDK1 can lead to cancer and other disorders such as inflammation,arthritis,diabetes,cardiovascular,and neurological diseases.The binding of ligand to the PIF pocket of PDK1 causes the allosteric effect to PDK1.SBF1 has been proved to have anti-growth effects to melanoma and cervical cancer cells in vitro and in vivo.In this work,we studied the binding stability,affinity,and key residues of the PIF pocket of PDK1 to SBF1~-(the hydrolyzed product of SBF1)binding by means of molecular docking,MD simulations,MM/PBSA,free energy decomposition,alanine scanning,umbrella sampling etc.,and compared with its known low-molecular-compound activators(PS48,PS172,PS182,and PS210).We calculated 2D PMF profile for SBF1~-and PDK1 binding by defining?B end-to-end distance and hinge motion as reaction coordinates via multistate Bennett acceptance ratio(MBAR)method.These results indicate that SBF1~-is a potential allosteric inhibitor of PDK1 targeting the PIF-binding pocket and can serve as a new scaffold template for the design of new drugs targeting PDK1 in the future.The last,but not the least,system we studied is murine cytomegalovirus(MCMV),which is a selected huge system(molecular weight for capsid is at mega-Da level)for virus system studies.Herpesviruses contain a wide range of virus types and can cause latent and lifelong infection to the host.Thus it is a very crucial system and was acquiring ascending attentions in recent years.The drug design to herpesvirus infections was impeded due to the lack of high resolution structures and the wise selection of drug target among hundreds types of herpesvirus proteins.Human cytomegalovirus(HCMV)infection can cause birth defects and life-threatening complications in immunosuppressed patients.The study of HCMV infection can not be conducted in human host,thus many related studies are performed using animals,mostly based on the mouse model with MCMV.Previous studies focused on infection mechanisms and immunogenic responses,little on structures,leaving a significant knowledge gap in such efforts.We thus resolved the icosahedral structure of MCMV at5?resolution by cryoEM and improved it to 3.6-3.8?resolution by localized reconstruction method,then further built atomic models.By comparison,MCMV and HCMV share highly similar capsid structures(except for the N-anchor domain of Tri1proteins in MCMV and HCMV),but have distinct capsid-associated tegument(pp150)binding patterns:(1)The pp150 in MCMV exists as dimer,but as dimer and monomer in HCMV;(2)No pp150 binding was observed in the triplex Tc regions in MCMV,but three group-of-three pp150s in HCMV;(3)Significant more inter-molecular interactions between pp150-pp150 dimer were observed in MCMV than in HCMV.The rigidity of pp150 dimer in MCMV and the longer distance between pp150 and its neighboring SCP explain the absence of pp150 in triplex Tc regions.pp150-deletion study suggests pp150 in MCMV is important but not essential for the virus replication in vitro.In contrast,pp150 is essential in HCMV.These results thus invalidate targeting pp150,but lend support to targeting capsid proteins(e.g.SCP),when using MCMV as a model for HCMV infection studies.As a summary,we studied three vital biomacromolecular systems at theoretical and experimental levels to unveil the relationship between their structures and functions.The conclusions can be drawn as following:(1)Molecular dynamic simulation is a useful and effective tool in the study of structure-function relationship for protein systems at medium size.But if long time scale is needed to realize the protein function,we can use some available methods to accelerate the MD processes,such as:umbrella sampling,replica-exchange MD(REMD),integrated tempering sampling(ITS),meta-dynamics,accelerated MD,steered MD,targeted MD.(2)The structures of carbohydrate are flexible and having lots of electrostatic effects.Current force fields remain problematic when describing their structures.There are many efforts that have being dedicated to continuously develop a reliable carbohydrate-specific force field.QM/MM method might be an easier solution but the optimization the description between protein and carbohydrate is required.(3)Once the proper reaction coordinates are discovered,umbrella sampling and MBAR methods can be used as effective tools to study allosteric regulation effects.(4)Localized reconstruction method is very effective while dealing with Ewald-sphere effect and focus gradient issues caused by giant virus particles during the cryoEM image acquirement,thus can improve the resolution for localized sub-particles.Molecular dynamic simulation remains challenging when coping with such huge systems,but multiscale simulation could be a solution in the future.The drug design based on the available herpesvirus structures would be a research trend in the future. |
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