Mechanism Of Cis-peptide Bond Formation And Its Functional Effects In Model Protein

The protein main skeleton chain can not rotate freely because the peptide bond-CO-NH-is considered to have a partially rigid property,and it forces the dihedral angle(?)to rotate only near 0° or 180°.When the ? is 0°,it is called cis-peptide bond,while when the ? is 180°,it is called trans-peptide bond.The conformation of the trans peptide bond is relatively stable,while the conformation of the cis-peptide bond is relatively unstable,so in general,the peptide bond on the main skeleton of the protein presents a trans conformation,while the cis-peptide bond occurs at a low frequency.Conversion between the trans-peptide bond and the cis-peptide bond formed by any two amino acids(peptide bond cis-trans isomerism)has a definite incidence of cis-peptide bonds(ICPB),and protein conformation and function are related to the ICPB value.Amino acid substitution mutation can lead to the change of ICPB value of local sites of protein molecules,which leads to the corresponding protein conformation and function changes in organisms.Therefore,it is of great significance to explore the occurrence and distribution ICPB in different peptide bonds on the backbone of protein backbone to reveal the relationship between protein structure and function.Usually,the ICPB value is small,and the isomerization of peptide bonds is a dynamic process.It is difficult to capture the conformation of cis-peptide bonds by conventional methods such as X-ray diffraction crystal technique,nuclear magnetic resonance,scanning 3D electron microscope and so on.Therefore,in this paper,we first theoretically derive the functional relationship of the torsional angle(?)of the peptide bond on protein backbone with the distance(d)between the adjacent C? atoms.The non-redundant data set is established by using PDB database,from which the cis peptide bond data are extracted,the ICPB values of different peptide bonds are counted and the rules of ICPB of different amino acid residues are summarized.Then,the molecular structure model of 20 dipeptides was created by molecular simulation.The dynamics simulated the change of free energy in the cis-trans isomerization process of peptide bonds and analyzed the variation law between them and their respective ICPB.Finally,using ?-catenin as model proteins,we constructed ?-catenin(Xaa246-P247)expression vectors with different ICPB values by point mutation method and introduced these vectors into Pin1 expression-positive hepatoma carcinoma cell lines(hep G2)to observe the interaction between different ?-catenin(Xaa246-P247)molecules and APC?E-cadherin,and the sub-localization levels of different(Xaa246-P247)molecules in hep G2,to investigate the effect relationship between the ICPB value of the ?-catenin(Xaa246-P247)site and the localization of ?-catenin subcells.The main findings are as follows:(1)In our data set,cis-peptide bonds account for 0.33% of the total number of peptide bonds,and cis-peptide bonds are mostly found on peptide bonds associated with proline(proline,P),where Xaa-P cis-peptide bonds account for 73% of the total number of cis-peptide bonds.The different Xaa species have different effects on the incidence of Xaa-P cis-peptide bonds,with relatively high ICPB values when Xaa are aromatic amino acids such as tyrosine(Y)and tryptophan(tryptophan).When Xaa are residues such as leucine(L),methionine(M),the ICPB value is relatively low.Except for a very small fraction of cis-peptide bonds distributed on residue fragments of proline-tryptophan,tryptophan-alanine,cysteine-cysteine,glycine-glycine,cis-peptide bonds rarely appear on non-proline peptide bonds(Xaa-Xn P)in general.Judging from the site where cis-peptide bonds occur,they generally occur in the flexible region of protein second structure,that is,C end of ?-helix,N end of ?-fold and irregular curl.(2)The 20 peptide bonds have basically achieved the minimum free energy in the trans configuration and are in a stable state with dihedral angles in the interval [-180°,-170°] or [170°,180°].A minimum of free energy is mostly distributed in interval [0,17]kcal/mol.Most of the dihedral angles of the minimum free energy of cis-peptide bonds are distributed in the interval [10°,25°],corresponding to the free energy value in interval [1,49] kcal/mol.Overall the free energy value of the cis peptide bond is greater than the free energy value of the trans peptide bond.Most of the energy peaks of 20 peptide bonds appear in the dihedral angles of [-110°,-60°] and [80°,110°],and the corresponding free energy values are distributed in the intervals [25,55] kcal/mol and[20,40] kcal/mol.Especially,there is a big gap in the free energy of E-P,P-P,N-P,Q-P.From the results of molecular simulation,the ICPB value of peptide bond is related to its cis-trans isomerism energy difference and energy barrier,which is the result of the interaction of amino acid residue structure,electrical property,polarity and so on.The ICPB values reflect the combined effect of these factors.(3)Our experimental results show that changes in Xaa246-P247 ICPB values on?-catenin molecules are responsible for changing interactions with ?-catenin/APC??-catenin/E-cadherin and ultimately affect subcellular localization of the molecules in the hep G2.There is a certain effect relationship between the ICPB value of Xaa246-P247 sites on ?-catenin molecules and the subcellular localization in hep G2cells(eukaryotic cells).The results of this study are of great significance to reveal the cis-trans isomerism law of protein peptide bond and its regulation of life activity.Furthermore,because the folding and de-folding process of proteins is also related to the cis-trans isomerization of peptide bonds,the ICPB law can also predict the structure of proteins,guide the synthesis of new functional proteins and the design of new polypeptide drugs.