Study On The Self-binding Peptide-mediated Conformation,Activity And Function Of Peroxisome Proliferator-activated Receptors

Peroxisome proliferators-activated receptors(PPARs)are a class of ligand-induced nuclear receptor transcription factors that can be activated via ligand binding,including three subtypes: PPARα,PPARβ and PPARγ,in which the PPARγ has been most documented.PPARγ plays an important role in the regulation of insulin resistance and glucose metabolism,and is one of the well-established druggable targets in the design of anti-diabetic drugs.Studies on the structure and function of the ligand binding domain(LBD)of PPARs showed that the molecular behavior and dynamics of the C-terminal helix 12(H12)of LBD plays an essential role in the regulation of PPAR activity and funcation.The C-terminal helix(H12)is a short peptide fragment located inside the monomer protein,connected to the parent protein in the primary sequence through a flexible linker,and has an independent structure and function.It can dynamically and specifically recognize,bind and dissociate with a specific region of the parent protein receptor,regulate cell protein-protein interactions,and weave complicated cell signal networks.Previously,we called the new short peptides with this characteristic "self-binding peptides".However,since it is a great challenge to prepare crystal structure for the transition state of H12 regulation between PPAR activation and inactivation,the structure,function and dynamics behavior of H12 still remain largely unexplored to date.In this study,we have carried out a systematic work on the H12 self-binding peptide of PPARs.Firstly,the primary sequence of H12 peptide was defined in its crystal structure,and its molecular behavior involved in both the protein folding and binding was elucidated.It shows a typical characteristic of activation and inactivation in different ligand binding modes.Secondly,we analyzed the various subtypes of PPARs in the protein data bank(PDB)database as well as the crystal structures of protein-protein and protein-small molecule complexes that bind different ligands,and selected a number of representative complex systems to perform atomistic molecular dynamics simulations and binding energy analyses,in order to compare their thermodynamic properties and dynamic behaviors in the protein context of PPAR LBD domain in complex with agonists,antagonists and inverse agonist ligands.The binding mode and energetic properties with different types of agonists,antagonists and inverse agonists were also characterized in detail.Next,we rationally designed several specific mutants of H12 self-binding peptide in three PPAR subtypes,and calculated and compared their binding modes and energies in different subtypes,which can further clarify the receptor-ligand binding pocket of H12self-binding peptides.Finally,we selected several protein-peptide complex systems that partially meet the concept of "self-binding peptides" in the protein data bank(PDB).Through dynamics simulation and energetic analysis we explored how these self-binding peptides to function as the "induced fit" or "conformational selection" model,and then compared them with the current H12 self-binding peptide in PPARs to promote our findings.In this subject,after a series of studies and comparisons,we came to the following conclusions:(1)The H12 self-binding peptide is highly flexible and exhibits large intrinsic disorder.Its N-terminus is connected to its parent protein of PPAR LBD domain,with considerable variation in its C-terminus,where would be the key region involved in its binding and unbinding.(2)The H12 is a typical self-binding peptide that represents a short peptide fragment within the PPAR LBD domain to fulfill biological functions by dynamically binding to/unbinding from the same domain.The peptide possesses a large intrinsic disorder in unbinding state,but is highly structured upon binding to the domain,which is so-called as folding-upon-binding.(3)The energy of LBD–H12 interaction system primarily comes from the polar electrostatic forces between them;the electrostatic contribution would not be influenced substantially when replacing the H12 with other mutants.The binding/unbinding of H12 to/from LBD has only a minor effect on the whole protein system.The interaction mechanism between the LBD and H12 can be considered as "induced fit" or "conformational selection".The dynamic behavior of different H12 mutants is roughly consistent but their binding energies are slightly different.As compared to electrostatic effect the van der Waals force plays an increased role in smaller systems.