Helix 3 is necessary and sufficient for prion protein’s anti-Bax function

To identify the structural elements of the prion protein (PrP) necessary for its protective function against Bcl-2 associated protein X (Bax), we performed structure–function analyses of the anti-Bax function of cytosolic PrP (CyPrP) in MCF-7 cells. Deletions of 1, 2, or 3 N-terminal Bcl-2 homology domain 2-like octapeptide repeats (BORs), but not deletion of all four BORs, abolish CyPrPs anti-Bax function. Deletion of α-helix 3 (PrP23–199) or further C-terminal deletions of α-helix 1 and 2, and β-strand 1 and 2 (PrP23–172, PrP23–160, PrP23–143, and PrP23–127) eliminates CyPrPs protection against Bax-mediated cell death. The substitution of helix 3 amino acid residues K204, V210, and E219 by proline inhibits the anti-Bax function of CyPrP. The substitution of K204, but not V210 and E219, by alanine residues also prevents CyPrPs anti-Bax function. Expression of PrPs helix 3 displays anti-Bax activity in MCF-7 cells and in human neurons. Together, these results indicate that although the BOR domain has an influence on PrPs anti-Bax function, the helix 3 is necessary and sufficient for the anti-Bax function of CyPrP. Identification of helix 3 as the structural element for the anti-Bax function thus provides a molecular target to modulate PrPs anti-Bax function in cancer and neurodegeneration.