Crystal Structure Of Chiloscyllium Plagiosum TBC1D15 Protein

TBC domain(Rab-GAP)is a highly conserved amino acid sequence that contains about 200 residues,almost all eukaryotes have the TBC domain protein.TBC1D15(TBC1 domain family member 15)belongs to TBC domain family with varieties of physiological functions,which plays an important role in biological regulation.According to the previous work,we find that TBC1D15 is a conserved protein serving as GTPase-activating protein especially to Rab7.TBC1D15 can modulate the lysosomal morphology after adjusting Rab7.Besides,it is also important to maintain the dependence of growth factors.TBC1D15 of Chiloscyllium plagiosum(Shark-TBC1D15)was first found in regenerated shark liver that contains 4213 bp gene sequence.The N-terminus of Shark-TBC1D15 has APSL(Active Peptide from Shark Liver)domain related to liver regeneration in Chiloscyllium plagiosum.APSL also has many other functions,such as reducing the blood glucose level in mice with type 2 diabetes,being a noteworthy immunomodulatory and inhibiting lipid peroxidation activity.Our group found that Shark-TBC1D15 was the GTPase-activating protein(GAP)for Rab7 a through GAP assays in vitro.Although we had determined the GAP activity of Shark-TBC1D15 in vitro,the catalytic mechanism is not clear.Until now,there is no TBC1D15 structural mode to help us elucidate the mechanism,so we hope using the crystallographic method to solve the structures of Shark-TBC1D15 and Shark-TBC1D15·Rab complex.Using bioinformatics method to analyze the full protein sequence of TBC1D15(Shark/ Homo/ Sus/ Mus),we designed four different truncations for this four proteins,among which the best proteins were screened after protein expression,purification and HPLC identification.Later,through crystallization experiments,the suitable crystallization conditions were screened.After optimizing the crystallization conditions,the protein crystals which have high resolution were obtained.The results showed that Sus GAP-2,Shark GAP-2,Homo GAP-2 and Mus GAP-2 all have high expression,Shark GAP-2 and Sus GAP-2 crystals were obtained through crystallization.After optimizing the crystallization conditions and collecting the diffractive data,the crystal structures of Shark GAP-2 and Sus GAP were solved at 2.5 ? and 2.8 ? resolution,respectively.Comparing the two protein structures,their structures were quite similar,which also demonstrated the conservatism of TBC1D15 in evolution.Besides,comparing the structures with yeast Gyp1 p and TBC1D1,the Shark and Sus TBC1D15 GAP domains have similar overall framework about 16 ?-helices and no ?-sheet elements,but there also have several difference between them.The results indicate that the structures of Shark GAP-2 and Sus GAP-2 are two new protein structures,which is also a supplement for the structures of TBC domain family.In addition,GAP assays had been conducted to examine the substrate-preference of Shark GAP-2 and Sus GAP-2 on Rab7 a and Rab11 a.The results show that Shark GAP-2 and Sus GAP-2 both have catalytic activity on Rab11 a and Rab7 a which hydrolyze GTP to GDP.According to the structures of Shark GAP-2 and Sus GAP-2,mutation experiment was conducted about arginine and glutamine which locate on catalytic motifs: Shark GAP-2(R437A,R437 K,Q474A)and Sus GAP-2(R400A,R400 K,Q437A).After GAP assays,when the arginine and glutamine were changed to alanine or lysine,the activity of Shark GAP-2 and Sus GAP-2 would be lost.It indicates that arginine and glutamine are key amino acid for catalytic reaction,and one of the two residues is mutated,which will lead to the loss of GAP activity.Finally,we tried to get the complex structures of Shark GAP-2/ Sus GAP-2 with Rab7a/ Rab11 a through coexpression and tandem expression.However,the results showed that coexpression could not get the objective proteins,while tandem expression could get the proteins which we expect.Unfortunately,we could not get the protein crystals of the tandem proteins(Shark1215+3GSA+Rab7a/Rab11a?Sus2010+3GSA+Rab7a/Rab11a).