Conformational Changes And Kinetics Of Phosphorylation Induced Bcl-2 Protein

Bcl-2 protein is a tumor target protein.Its BH domains have been obtained by structural biologists,but the structure of its loop region(residues 34-92)has not yet been resolved.It is worth noting that the Bcl-2 protein is able to phosphorylate single or multiple sites of Thr69,Ser70,Ser87 in the loop region.Recent studies have shown that cell lines that highly express phosphorylated Bcl-2 protein are resistant to FDA-approved Bcl-2 inhibitor anticancer drugs ABT-199 and its derivatives.These findings all indicate that although the loop region is spatially distant from the BH domain,its structural changes can regulate the function of the Bcl-2 protein.However,it is still unclear whether the phosphorylation of Bcl-2 affects the domains and thus leads to a change in function.The analysis of the Bcl-2 molecular structure containing the loop region will solve such problems,for example,how the phosphorylation modification regulates the interaction between Bcl-2 and the ligand,and how the phosphorylation of Bcl-2 leads to the mechanism that the existing anticancer drugs produce resistance to the phosphorylated Bcl-2(pBcl-2),and thus helps us to design inhibitors that target pBcl-2.At present,the method of NMR or X-Ray crystal diffraction can not analyze the structure of Bcl-2 protein containing loop region,and the computer molecular simulation technology has been an effective means to guide and assist the experiment for more than 30 years.We have homologously modeled Bcl-2 molecules by computational methods and performed full-atom molecular dynamics simulation(MD)on Bcl-2.Through six groups of long-term simulations of 200 ns,we found that Bcl-2 undergoes a conformational change in its BH3 groove after phosphorylation,while the BH3 groove is a common domain of the Bcl-2 family and is also the interface between the family members.In addition,through the principal component analysis of the MD simulation,it was found that the phosphorylation site in the loop region is closely related to the movement of the BH3 groove.MD simulations revealed that phosphorylation promoted a wide range of conformational changes in ?3 and the end positions of ?2 of Bcl-2.It was found that the change of BH3 groove conformation directly affects the affinity of Bcl-2 and ligand through binding free energy analysis of Bcl-2 and pBcl-2 with ligands by MM-GBSA method.It was also found that the binding free energy of Bcl-2_Bax BH3 increased after phosphorylation of Bcl-2 protein,and the binding free energy between Bcl-2 and Bim BH3 and ABT-199 was weakened.This result was also confirmed by biological experiments.Decomposition of binding free energy residues identified the key amino acid residues on the BH3 groove interface.After further comparing the contribution of these key residues in the Bcl-2 and pBcl-2 complexes to the binding energy,the key BH3 groove interface residues that modulate the anti-apoptotic function of Bcl-2 were identified.It also revealed the reason why Bcl-2 hyperphosphorylated tumor cells were resistant to ABT-199.