Structural Basis for the Dynamic Behavior of a Family of Disordered Proteins

Abstract

Intrinsically disordered proteins (IDPs) are characterized by a lack of stable tertiary structure and sample a dynamic range of conformations. When binding, IDPs often transition from a disordered to ordered state, with helices being the most common conformation adopted (1). The p53 tumor suppressor protein is an IDP that transitions from an unfolded (disordered) to folded (ordered) state upon binding. The p53 transactivation domain (TAD) forms an alpha helical structure when it binds to either the 70 kDa subunit of replication protein a (RPA70) or the murine double minute 2 protein (MDM2). We analyzed chemical shifts of RPA70 after titrating with either human p53 TAD, or the canine p53 TAD homologue using 15N-1H HSQC NMR experiments. Human p53 TAD appears to bind to RPA70 with a higher affinity than canine p53 TAD. Furthermore, canine p53 TAD is more dynamic than human p53 TAD, which is consistent with the titration results. There are two mutations in the canine p53 MDM2 binding site, D21E and K24N. The aspartic acid and lysine residues at positions 21 and 24 respectively, stabilize the helix of human p53 when bound to MDM2. D21E and K24N point mutants were created in human p53TAD using site directed mutagenesis. We are currently investigating how these mutants affect the interaction with MDM2. 1. Cheng Y, LeGall T, Oldfield CJ, Mueller JP, Van YY, Romero P, et al. Rational drug design via intrinsically disordered protein. Trends Biotechnol. 2006;24(10):435-42.