Study Of The Binding Mechanism Between The Tumor Suppressor APC And Amer1

APC protein is a negative regulator in Wnt signal pathway, which contains2843residues and owns multi-domains. Its N-terminal contains a highly conservative ARMdomain, which can bind number of proteins to play the role. Amer1which has nosignificant functional domains after the Bio-information analysis can bind toAPC-ARM domain by three sites reported early. They are A1(280-368), A2(380-531)andA3(716-834) respectively.We get a new conservative sequence of Amer1365-375by the BLAST andAlignment, which will becomes the forth site to bind with APC-ARM domain.Actually Tanneberger, K ignored the site by dividing the Amer1to three parts, and thesequence Amer1325-335is the first site to bind with APC–ARM domain (the datawill be published soon). We found the missed site and confirmed their combination bythe GST-Pulldown and ITC methods. Fortunately we got the crystal of complexAPC-ARM with the Amer1365-375and solved their structure, and the way ofcombination is similar to theA1,A2, Asef and Sam68binding with APC-ARM.We tried to get the crystal of complex APC-ARM with the A3. Because of theinaccurate position, so we intend to find the conservative sequences by BLAST andAlignment. Based on the result, we constructed the recombinant plasmid, expressedand purified the GST-tag proteins, such as Amer1716-834,766-823,766-777,786-823,766-823△778-794. After a series of GST-Pulldown and ITC experiments,we confirmed that the Amer1766-823△778-794is the shortest sequence which can bind to APC-ARM domain, whereas the sequence of Amer1766-777and Amer1786-823can neither bind to APC-ARM domain. That means the combination of A3with APC-ARM domain is different from the way of A1, A2and A4. The three latersequences can bind to APC-ARM domain by the nearby key amino acids. Weassumed that the N-terminal and C-terminal sequences formed a certain structurewhich plays a key role to bind with APC-ARM domain, and the middle residues, suchas778-794are not necessary for the combination.We tried many methods to purify the complex APC-ARM with Amer1766-823△778-794, which include the further purification after the digestion bythrombin, and digestion of the recombinant proteins APC-ARM-TEV-Amer1740/765-823consequently followed by FPLC. While we still cannot get the crystal ofcomplex APC-ARM with the A3after about one thousand conditions have been tried.The Amer1766-823△778-794is not the optimal sequence for the crystallization, andwe need further experiments to locate a more accurate sequence which binds withAPC-ARM.