Spectroscopic characterization of the complex between azurin and p53 transactivation domain

Recent reports have shown that the bacterial redox protein azurin can enter into cancer cells and induce apoptosis by stabilizing p53. The formation of a complex between the two proteins has been demonstrated, but little is known about binding features. For the first time, we show here that azurin binds to the N-terminal region of p53 with a dissociation constant in the 5-10 ?M range. Trp phosphorescence lifetime measurements revealed conformational changes of azurin induced by the interaction with p53(1-63). Acrylamide quenching of Trp phosphorescence also indicated a significant increase of the overall flexibility of azurin upon binding to p53(1-63). No change of the fluorescence emission of p53(1-63) was detected in the presence of azurin. The latter finding suggests that W23 of p53 is not directly involved in domain binding to azurin, indicating that the binding site is distinct from that of MDM2. The present results may assist the design of novel cancer treatments based on p53 stabilization by azurin.