A quantitative systems approach to define novel effects of tumour p53 mutations on binding oncoprotein MDM2

Abstract

[EN]Understanding transient protein interactions biochemically at the proteome scale remains a long‐standing challenge. Current tools developed to study protein interactions in high‐throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self‐assembling protein microarrays produced by freshly translating full‐length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label‐free platform that measures many protein interactions in real‐time allowing the determination of the KDs and rate constants. The developed novel NAPPA‐SPRi technique showed excellent abil-ity to study protein‐protein interactions of clinical mutants of p53 with its regulator MDM2. Fur-thermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin‐ 3, currently in clinical practice, which usually disrupts the p53‐MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg‐ 273‐Cys, Arg‐273‐His, Arg‐248‐Glu, Arg‐280‐Lys), on the structural domain (His‐179‐Tyr, Cys‐176‐ Phe), on hydrophobic moieties in the DNA binding domain (Arg‐280‐Thr, Pro‐151‐Ser, Cys‐176‐ Phe) and hot spot mutants (Gly‐245‐Cys, Arg‐273‐Leu, Arg‐248‐Glu, Arg‐248‐Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities.